Thermophilic DNA polymerase mutants

ABSTRACT

This disclosure relates to thermophilic family B DNA polymerases comprising a neutral amino acid residue at a certain position near the C-terminus of the catalytic domain, which corresponds to a position occupied by a basic amino acid residue in wild-type Pfu polymerase. Related uses, methods, and compositions are also provided. In some embodiments, the polymerases comprise a 3′-5′ exonuclease domain and/or a sequence non-specific dsDNA binding domain.

CROSS-REFERENCE

This application is a continuation application under 35 U.S.C. § 120 ofpending of U.S. application Ser. No. 15/405,574 filed Jan. 13, 2017,which claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 62/279,404, filed Jan. 15, 2016. The entire contents ofthe aforementioned applications are incorporated by reference herein.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2016-12-30_01158-0003-00US_ST25.txt” created on Dec. 30, 2016, which is332,338 bytes in size. The information in the electronic format of thesequence listing is incorporated herein by reference in its entirety.

This disclosure relates to the field of thermophilic DNA polymerasemutants, including methods, uses, and compositions thereof.

Thermophilic DNA polymerases are commonly used in biotechnology andmolecular biology applications, including nucleic acid synthesistechniques such as amplification (e.g., polymerase chain reaction, orPCR), which involves cycles of alternating denaturation and primerannealing and extension. Thermophilic DNA polymerases are resistant toinactivation by high temperatures and so are compatible with thermaldenaturation steps. DNA polymerases comprise a catalytic domain thatextends a 3′ terminus of a DNA strand in a template-dependent manner.DNA polymerases can also comprise an exonuclease domain, such as a 3′ to5′ exonuclease domain. Such an exonuclease domain can reduce thefrequency of misincorporation by removing mismatched nucleotides fromthe 3′ end of a nascent DNA strand. Certain artificial DNA polymerasesfurther comprise a sequence non-specific double-stranded DNA (dsDNA)binding domain. The presence of this domain can improve performance ofthe enzyme with respect to various parameters, including processivity,sensitivity, and yield.

Nucleic acid amplification can permit rapid detection of a targetnucleic acid sequence and/or provide sufficient quantities of a samplefor further analysis or manipulation, such as sequencing, cloning,restriction digestion, hybridization, ligation, mutagenesis,recombination, etc. Two key parameters of amplification are sensitivityand yield. Improving the sensitivity reduces the minimum amount of atarget needed to produce a detectable product. Improving the yieldincreases the amount of product that results from a reaction, or reducesthe amount of time and/or reagents necessary to obtain a given amount ofproduct.

Samples may be refractory to amplification or may decrease sensitivityand/or yield if they contain nucleic acid synthesis inhibitors, whichmay occur naturally in the sample or may be introduced during earliersample processing steps. Examples of nucleic acid synthesis inhibitorsinclude polyanions such as heparin or xylan; anionic detergents such assodium dodecyl sulfate; and certain complex organic substances such ashumic acid, collagen, heme and heme-containing proteins, bile salts, andthe like. Thermophilic DNA polymerases with improved tolerance of suchinhibitors would reduce the need for purification and other sampleprocessing steps in advance of nucleic acid synthesis and reduce thefrequency of unsatisfactory synthesis reactions.

Certain polymerases such as the family B polymerases, includingPyrococcus furiosus (Pfu) DNA polymerase (see Kennedy et al., “TheMechanistic Architecture of the Thermostable Pyrococcus furiosus FamilyB DNA Polymerase Motif A and its Interaction with dNTP Substrate,”Biochemistry 2009 Dec. 1; 48(47): 11161-11168. doi:10.1021/bi9010122)and related polymerases, may benefit from mutations that increase yieldand/or sensitivity. In some instances, an A408S mutation has beenintroduced into family B polymerases in order to improve accuracy (i.e.,reduced error rate or increased fidelity), but with a detrimental impacton yield and/or sensitivity. It would be desirable to provide variantsof family B polymerases that have improved yield and/or sensitivity.Further, coupled with an A408S mutation, such variants may have improvedyield and/or sensitivity and also improved fidelity. It would also bedesirable to provide variants of such polymerases with improvedinhibitor resistance. Such polymerases could be suitable for use with abroader spectrum of samples and/or could reduce the need forpreprocessing in advance of nucleic acid synthesis reactions in whichhigh fidelity is desirable, such as for cloning, sequencing, geneconstruction, site-directed mutagenesis, etc.

Thus, there are needs for thermophilic DNA polymerases having increasedinhibitor tolerance and/or the capability to provide increased yieldand/or sensitivity. Provided herein are polymerases and related methodsand compositions that can solve these needs and/or provide otherbenefits.

In some embodiments, the present disclosure provides thermophilic DNApolymerase mutants and methods of nucleic acid synthesis usingthermophilic DNA polymerase mutants. In some embodiments, a thermophilicDNA polymerase comprising a family B polymerase catalytic domain isprovided, the family B polymerase catalytic domain having an amino acidsequence in which the position corresponding to position 379 of SEQ IDNO: 6 is a neutral amino acid residue. In some embodiments, the family Bpolymerase catalytic domain has at least 80%, 85%, 90%, 95%, 98%, 99%,or 100% identity to the family B polymerase catalytic domain sequence ofa sequence selected from SEQ ID NOs: 6 to 10, 15 to 18, 25, 26, 33, 34,37, 38, 41, 42, and 45 to 48, wherein X is the neutral amino acidresidue and is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V,P, and G. In some embodiments, X is N or G. In some embodiments, theamino acid residue at the position of the amino acid sequence thataligns to position 379 of SEQ ID NO: 6 is a neutral amino acid residue.In some embodiments, the family B polymerase catalytic domain has atleast 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 6. Insome embodiments, the amino acid residue at the position of the aminoacid sequence that corresponds to position 25 of SEQ ID NO: 6 is aserine. In some embodiments, the family B polymerase catalytic domaincomprises a consecutive amino acid sequence of WQKTX, XQTGL, KTXQT,YQKTX, XQVGL, KTXQV, YQSSX, XQTGL, SSXQT, TGRVX, XKSLL, RVXKS, TGRSX,XRTLL, or RSXRT;

wherein X is a neutral amino acid residue; and wherein X is within 20residues of the C-terminus of the family B polymerase catalytic domain.In some embodiments, the family B polymerase catalytic domain is asubfamily B3 polymerase domain. In some embodiments, the neutral aminoacid residue is a polar neutral amino acid residue. In some embodiments,the neutral amino acid residue comprises an amide. In some embodiments,the neutral amino acid residue is selected from Q, N, H, S, T, Y, C, M,W, A, I, L, F, V, P, and G. In some embodiments, the neutral amino acidresidue is selected from Q and N. In some embodiments, the neutral aminoacid residue is Q.

In some embodiments, the thermophilic DNA polymerase comprises asequence non-specific double-stranded DNA-binding domain. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to a sequence selected from SEQ IDNOs: 53 to 62. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain is C-terminal to the family Bpolymerase catalytic domain. In some embodiments, the sequencenon-specific double-stranded DNA-binding domain is a 7 kD DNA-bindingdomain. In some embodiments, the sequence non-specific double-strandedDNA-binding domain is an Sso7d, Sac7d, or Sac7e domain.

In some embodiments, the thermophilic DNA polymerase comprises: (a) theconsecutive amino acid residues LDFRS, (b) the consecutive amino acidresidues FRSLY, or (c) the consecutive amino acid residues SLYPS,wherein the underlined serine residue is within 30 amino acid residuesof the N-terminus of the family B polymerase catalytic domain.

In some embodiments, the thermophilic DNA polymerase comprises a 3′ to5′ exonuclease domain. In some embodiments, the 3′ to 5′ exonucleasedomain is N-terminal to the family B polymerase catalytic domain. Insome embodiments, the 3′ to 5′ exonuclease domain is a DEDDy archaealexonuclease domain. In some embodiments, the 3′ to 5′ exonuclease domaincomprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or100% identity to SEQ ID NO: 63. In some embodiments, the 3′ to 5′exonuclease domain comprises an amino acid sequence having at least 90%,95%, 98%, 99%, or 100% identity to the 3′ to 5′ exonuclease domain of asequence selected from SEQ ID NOs: 1, 19, 23, 31, 35, 39, 43, 49, 51,52, and 76 to 79.

In some embodiments, the thermophilic DNA polymerase comprises an aminoacid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to asequence selected from SEQ ID NOs: 11 to 14, 19 to 22, 27 to 30, and 76to 79, wherein X is the neutral amino acid residue and is selected fromQ, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G. In some embodiments,X is N or G.

In some embodiments, the thermophilic DNA polymerase comprises an aminoacid sequence comprising (i) at least one difference at a positioncorresponding to position 15, 72, 93, 141, 143, 247, 265, 337, 385, 387,388, 399, 400, 405, 407, 410, 485, 542, 546, 593, or 595 of SEQ ID NO: 1or (ii) at least one missing residue corresponding to position 92, 93,94, or 381 of SEQ ID NO: 1. In some embodiments, the at least onemismatch or missing residue comprises at least one of:

(i) a missing residue corresponding to position 92 or 94 of SEQ ID NO:1;

(ii) a Q or R at the position corresponding to position 93 of SEQ ID NO:1;

(iii) an A at the position corresponding to position 141 of SEQ ID NO:1;

(iv) an A at the position corresponding to position 143 of SEQ ID NO: 1;

(v) an I at the position corresponding to position 337 of SEQ ID NO: 1;

(vi) a Q, S, N, L, or H at the position corresponding to position 385 ofSEQ ID NO: 1;

(vii) a P or S at the position corresponding to position 387 of SEQ IDNO: 1;

(viii) a P at the position corresponding to position 388 of SEQ ID NO:1;

(ix) a D at the position corresponding to position 399 of SEQ ID NO: 1;

(x) a G or D at the position corresponding to position 400 of SEQ ID NO:1;

(xi) an E at the position corresponding to position 405 of SEQ ID NO: 1;

(xii) an I at the position corresponding to position 407 of SEQ ID NO:1;

(xiii) an L or F at the position corresponding to position 410 of SEQ IDNO: 1;

(xiv) a T at the position corresponding to position 485 of SEQ ID NO: 1;

(xv) a P at the position corresponding to position 542 of SEQ ID NO: 1;

(xvi) an H at the position corresponding to position 546 of SEQ ID NO:1;

(xvii) a T at the position corresponding to position 593 of SEQ ID NO:1; or

(xviii) an S at the position corresponding to position 595 of SEQ ID NO:1.

In some embodiments, the thermophilic DNA polymerase has at least one ofthe following properties:

(i) capable of amplifying a 2 kb target from 40 ng of human genomic DNAtemplate in the presence of 0.2 μM heparin in a PCR; or

(ii) capable of amplifying a 2 kb target from 40 ng of human genomic DNAtemplate in the presence of 400 ng/μl xylan in a PCR;

wherein amplification is successful if product is detectable by agarosegel electrophoresis and ethidium bromide staining within 30 PCR cycles.

In some embodiments, the thermophilic DNA polymerase is bound to athermolabile inhibitor. In some embodiments, the thermolabile inhibitorcomprises an antibody, an Affibody®, an oligonucleotide, such as anaptamer, and/or a chemical modification.

In some embodiments, a method of in vitro nucleic acid synthesis isprovided, comprising contacting at least one primer and at least onetemplate with a thermophilic DNA polymerase provided herein in thepresence of at least one dNTP. In some embodiments, the thermophilic DNApolymerase is initially bound to a thermolabile inhibitor and the methodcomprises denaturing the inhibitor. In some embodiments, the methodfurther comprises amplification of the template. In some embodiments,the amplification comprises a PCR.

In some embodiments, a nucleic acid comprising a sequence encoding athermophilic DNA polymerase described herein is provided. In someembodiments, an expression vector comprising the nucleic acid isprovided. In some embodiments, an isolated host cell comprising thenucleic acid or the expression vector is provided. In some embodiments,a method of producing a thermophilic DNA polymerase described herein isprovided, comprising culturing at least one host cell comprising anucleic acid encoding the thermophilic DNA polymerase, wherein the atleast one host cell expresses the thermophilic DNA polymerase. In someembodiments, the method further comprises isolating the thermophilic DNApolymerase.

In some embodiments, compositions comprising thermophilic DNApolymerases described herein are provided. In some embodiments, thecomposition comprises at least one hot start inhibitor. In someembodiments, the composition comprises at least two hot startinhibitors. In some embodiments, each hot start inhibitor isindependently selected from an antibody, an Affibody®, anoligonucleotide and/or a chemical modification. In some embodiments, thecomposition comprises at least two antibodies. In some embodiments, thecomposition comprises an antibody and an oligonucleotide. In someembodiments, the oligonucleotide is an aptamer. In some embodiments, thecomposition comprises at least one antibody, and an Affibody® or anaptamer.

In some embodiments, the composition is a storage composition. In someembodiments, the composition comprises at least one protein stabilizer.In some embodiments, the protein stabilizer is selected from BSA,inactive polymerase, and apotransferrin. In some embodiments, thecomposition comprises a UTPase. In some embodiments, the compositioncomprises at least one buffering agent. In some embodiments, thebuffering agent is selected from acetate buffer, sulfate buffer,phosphate buffer, MOPS, HEPES and Tris-(hydroxymethyl)aminomethane(TRIS). In some embodiments, the composition comprises at least onemonovalent cationic salt. In some embodiments, the monovalent cationicsalt is selected from KCl and NaCl. In some embodiments, the compositioncomprises at least one stabilizer. In some embodiments, the stabilizeris selected from glycerol, trehalose, lactose, maltose, galactose,glucose, sucrose, dimethyl sulfoxide (DMSO), polyethylene glycol, andsorbitol. In some embodiments, the composition comprises at least onereducing agent. In some embodiments, the reducing agent isdithiothreitol (DTT). In some embodiments, the composition comprises atleast one divalent chelating agent. In some embodiments, the divalentchelating agent is EDTA. In some embodiments, the composition comprisesat least one detergent. In some embodiments, the detergent is anionic.In some embodiments, the detergent is cationic. In some embodiments, thedetergent is non-ionic. In some embodiments, the detergent iszwitterionic. In some embodiments, the composition comprises a detergentselected from Hecameg(6-O—(N-Heptylcarbamoyl)-methyl-α-D-glucopyranoside), Triton X-200,Brij-58, CHAPS, n-Dodecyl-b-D-maltoside, NP-40, sodium dodecyl sulphate(SDS), TRITON® X-15, TRITON® X-35, TRITON® X-45, TRITON® X-100, TRITON®X-102, TRITON® X-114, TRITON® X-165, TRITON® X-305, TRITON® X-405,TRITON® X-705, Tween® 20 and/or ZWITTERGENT®.

In some embodiments, the composition is an aqueous solution. In someembodiments, the composition is a lyophilized composition.

In some embodiments, the composition is a reaction composition. In someembodiments, the composition comprises at least one buffering agent. Insome embodiments, the buffering agent is selected from acetate buffer,sulfate buffer, phosphate buffer, MOPS, HEPES andTris-(hydroxymethyl)aminomethane (TRIS). In some embodiments, thecomposition comprises at least one monovalent cationic salt. In someembodiments, the monovalent cationic salt is selected from KCl and NaCl.In some embodiments, the composition comprises at least one divalentcationic salt. In some embodiments, the divalent cationic salt is MgCl₂or MnCl₂. In some embodiments, the composition comprises at least onedetergent. In some embodiments, the detergent is anionic. In someembodiments, the detergent is cationic. In some embodiments, thedetergent is non-ionic. In some embodiments, the detergent iszwitterionic. In some embodiments, the composition comprises a detergentselected from Hecameg(6-O—(N-Heptylcarbamoyl)-methyl-α-D-glucopyranoside), Triton X-200,Brij-58, CHAPS, n-Dodecyl-b-D-maltoside, NP-40, sodium dodecyl sulphate(SDS), TRITON® X-15, TRITON® X-35, TRITON® X-45, TRITON® X-100, TRITON®X-102, TRITON® X-114, TRITON® X-165, TRITON® X-305, TRITON® X-405,TRITON® X-705, Tween® 20 and/or ZWITTERGENT®. In some embodiments, thecomposition comprises at least one dNTP. In some embodiments, thecomposition comprises dATP, dGTP, dTTP, and dCTP. In some embodiments,the composition further comprises glycerol, DMSO, and/or ammoniumsulphate. In some embodiments, the composition comprises at least onedye. In some embodiments, the composition comprises at least one dyeselected from xylene cyanol FF, tartrazine, phenol red, quinolineyellow, zylene cyanol, Brilliant Blue, Patent Blue, indigocarmine, acidred 1, m-cresol purple, cresol red, neutral red, bromocresol green, acidviolet 5, bromo phenol blue, and orange G. In some embodiments, thecomposition comprises at least one agent that increases the density ofthe composition. In some embodiments, the composition comprises at leastone agent selected from PEG 4000 and/or sucrose. In some embodiments,the composition comprises at least one primer. In some embodiments, thecomposition comprises at least one nucleic acid template.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of PCR amplifications in which heparin waspresent at a series of concentrations from 0 to 0.3 μM and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain with (“762Q”) or without (“762K”) a neutral amino acid residue atthe position that aligns to position 762 of Pfu (SEQ ID NO: 1; aligningto position 379 of the Pfu catalytic domain (SEQ ID NO: 6)) and with(“408S”) or without (no 408 designation) a serine at the position thataligns to position 762 of Pfu (SEQ ID NO: 1; aligning to position 25 ofthe Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 2 shows a comparison of PCR amplifications in which xylan waspresent at a series of concentrations from 0 to 400 ng/μ1 and in whichthe polymerase comprised a family B thermophilic DNA polymerasecatalytic domain with (“762Q”) or without (“762K”) a neutral amino acidresidue at the position that aligns to position 762 of Pfu (SEQ ID NO:1; aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6))and with (“408S”) or without (no 408 designation) a serine at theposition that aligns to position 762 of Pfu (SEQ ID NO: 1; aligning toposition 25 of the Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 3 shows a comparison of PCR amplifications in which humic acid waspresent at a series of concentrations from 0 to 1 ng/μ1 and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain with (“762Q”) or without (“762K”) a neutral amino acid residue atthe position that aligns to position 762 of Pfu (SEQ ID NO: 1; aligningto position 379 of the Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 4 shows a comparison of PCR amplifications in which sodium dodecylsulfate (“SDS”) was present at a series of concentrations from 0 to0.016% or 0.2% (w/v) and in which the polymerase comprised a family Bthermophilic DNA polymerase catalytic domain with (“762Q”) or without(“762K”) a neutral amino acid residue at the position that aligns toposition 762 of Pfu (SEQ ID NO: 1; aligning to position 379 of the Pfucatalytic domain (SEQ ID NO: 6)).

FIG. 5 shows a comparison of PCR amplifications in which a 2 kb fragmentwas amplified from a series of amounts of human genomic DNA templatebetween 0 and 400 ng in a 20 μl PCR mixture using a polymerasecomprising a family B thermophilic DNA polymerase catalytic domain with(“762Q”) or without (“762K”) a neutral amino acid residue at theposition that aligns to position 762 of Pfu (SEQ ID NO: 1; aligning toposition 379 of the Pfu catalytic domain (SEQ ID NO: 6)) and with(“408S”) or without (no 408 designation) a serine at the position thataligns to position 762 of Pfu (SEQ ID NO: 1; aligning to position 25 ofthe Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 6A shows a comparison of PCR amplifications in which a 10 kbfragment was amplified from a series of amounts of bacteriophage lambdaDNA template between 0 and 200 ng in a 20μl PCR mixture using apolymerase comprising a family B thermophilic DNA polymerase catalyticdomain with (“762Q”) or without (“762K”) a neutral amino acid residue atthe position that aligns to position 762 of Pfu (SEQ ID NO: 1; aligningto position 379 of the Pfu catalytic domain (SEQ ID NO: 6)) and with(“408S”) or without (no 408 designation) a serine at the position thataligns to position 762 of Pfu (SEQ ID NO: 1; aligning to position 25 ofthe Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 6B shows a bar graph illustrating yield from amplification of a 10kb fragment from a series of amounts of bacteriophage lambda DNAtemplate using a polymerase comprising a family B thermophilic DNApolymerase catalytic domain with (“762Q”) or without (“762K”) a neutralamino acid residue at the position that aligns to position 762 of Pfu(SEQ ID NO: 1; aligning to position 379 of the Pfu catalytic domain (SEQID NO: 6)) and with (“408S”) or without (no 408 designation) a serine atthe position that aligns to position 762 of Pfu (SEQ ID NO: 1; aligningto position 25 of the Pfu catalytic domain (SEQ ID NO: 6)). FIG. 7Ashows a comparison of PCR amplifications of a 2 kb product in whichhuman genomic DNA template was present at a series of amounts from 0 to400 ng in a reaction volume of 20 μl and in which the polymerasecomprised a family B thermophilic DNA polymerase catalytic domainwithout (“408S 762K”) or with (“408S 762Q”) a neutral amino acid residueat the position that aligns to position 762 of Pfu (SEQ ID NO: 1;aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6)) andwith a serine at the position that aligns to position 762 of Pfu (SEQ IDNO: 1; aligning to position 25 of the Pfu catalytic domain (SEQ ID NO:6)).

FIG. 7B shows a comparison of PCR amplifications of a 5 kb product inwhich bacteriophage lambda DNA template was present at a series ofamounts from 0 to 200 ng in a reaction volume of 20 μl and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain without (“408S 762K”) or with (“408S 762Q”) a neutral amino acidresidue at the position that aligns to position 762 of Pfu (SEQ ID NO:1; aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6))and with a serine at the position that aligns to position 762 of Pfu(SEQ ID NO: 1; aligning to position 25 of the Pfu catalytic domain (SEQID NO: 6)).

FIG. 8 shows a comparison of PCR amplifications of a 20 kb product inwhich bacteriophage lambda DNA template was present at a series ofamounts from 0 to 100 ng in a reaction volume of 20 μl and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain without (“408S 762K”) or with (“408S 762Q”) a neutral amino acidresidue at the position that aligns to position 762 of Pfu (SEQ ID NO:1; aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6))and with a serine at the position that aligns to position 762 of Pfu(SEQ ID NO: 1; aligning to position 25 of the Pfu catalytic domain (SEQID NO: 6)).

FIG. 9 shows a comparison of PCR amplifications of a 20 kb product inwhich Escherichia coli genomic DNA template was present at a series ofamounts from 0 to 40 ng in a reaction volume of 20 μl and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain without (“408S 762K”) or with (“408S 762Q”) a neutral amino acidresidue at the position that aligns to position 762 of Pfu (SEQ ID NO:1; aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6))and with a serine at the position that aligns to position 762 of Pfu(SEQ ID NO: 1; aligning to position 25 of the Pfu catalytic domain (SEQID NO: 6)).

FIG. 10 shows a comparison of PCR amplifications of a 7.5 kb product inwhich human genomic DNA template was present at a series of amounts from0 to 400 ng in a reaction volume of 20 μl and in which the polymerasecomprised a family B thermophilic DNA polymerase catalytic domainwithout (“408S 762K”) or with (“408S 762Q”) a neutral amino acid residueat the position that aligns to position 762 of Pfu (SEQ ID NO: 1;aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6)) andwith a serine at the position that aligns to position 762 of Pfu (SEQ IDNO: 1; aligning to position 25 of the Pfu catalytic domain (SEQ ID NO:6)).

FIGS. 11A through 11B show a multiple amino acid sequence alignment ofThermococcus litoralis (“Tli”; SEQ ID NO: 31), (“Tsp9N7”; SEQ ID NO:49), Thermococcus gorgonarius (“Tgo”; SEQ ID NO: 39), Thermococcuskodakarensis (“Tko”; SEQ ID NO: 43), Pyrococcus furiosus (“Pfu”; SEQ IDNO: 2), and Deep Vent (“DP”; SEQ ID NO: 23) polymerases, in which theposition corresponding to position 408 of Pfu (SEQ ID NO: 1) is markedwith an asterisk (*) and the position corresponding to position 762 ofPfu (SEQ ID NO: 1) is marked with a pound (#). The positioncorresponding to position 762 of Pfu is indicated as “X” in each aminoacid sequence of the sequence alignment. X may be selected from Q, N, H,S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X isselected from Q and N. In some embodiments, X is Q.

FIG. 12 shows a multiple amino acid sequence alignment of the catalyticdomains of Thermococcus litoralis (“Tli”; SEQ ID NO: 33), (“Tsp9N7”; SEQID NO: 47), Thermococcus gorgonarius (“Tgo”; SEQ ID NO: 41),Thermococcus kodakarensis (“Tko”; SEQ ID NO: 45), Pyrococcus furiosus(“Pfu”; SEQ ID NO: 7), and Deep Vent (“DP”; SEQ ID NO: 25) polymerases,in which the position corresponding to position 408 of Pfu in thefull-length polymerase (SEQ ID NO: 1; corresponding to position 25 ofthe Pfu catalytic domain (SEQ ID NO: 6)) is marked with an asterisk (*)and position corresponding to position 762 of Pfu in the full-lengthpolymerase (SEQ ID NO: 1; corresponding to position 379 in the Pfucatalytic domain (SEQ ID NO: 6)) is marked with a pound (#). Theposition corresponding to position 762 of Pfu (SEQ ID NO: 1; position379 in the Pfu catalytic domain (SEQ ID NO: 6)) is indicated as “X” inthe sequence alignment. X may be selected from Q, N, H, S, T, Y, C, M,W, A, I, L, F, V, P, and G; in some embodiments, X is selected from Qand N. In some embodiments, X is Q.

DETAILED DESCRIPTION

This description and exemplary embodiments should not be taken aslimiting. For the purposes of this specification and appended claims,unless otherwise indicated, all numbers expressing quantities,percentages, or proportions, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about,” to the extent they are not already somodified. Accordingly, unless indicated to the contrary, the numericalparameters set forth in the following specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” and any singular use of anyword, include plural referents unless expressly and unequivocallylimited to one referent. As used herein, the term “include” and itsgrammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

The term “nucleic acid synthesis” refers to template-directed synthesisof a nucleic acid strand using a polymerase enzyme. Nucleic acidsynthesis includes all such template-directed nucleic acid synthesis bya polymerase, including, but not limited to, amplification, PCR, endpoint PCR (epPCR), real time or quantitative PCR (qPCR), one-stepRT-PCR, sequencing, etc.

As used herein the terms “amplify”, “amplifying”, “amplification” andother related terms include producing multiple copies of an originalbiomolecule, such as a nucleic acid. In some embodiments, nucleic acidamplification produces multiple copies of an original nucleic acidand/or its complement (e.g., target nucleic acid, also referred to as atarget polynucleotide), where the copies comprise at least a portion ofthe template sequence and/or its complement. Such copies may besingle-stranded or double-stranded.

A “template” or “template nucleic acid” or “template polynucleotide”refers to a polynucleotide that comprises the polynucleotide sequence tobe amplified. In some embodiments, the polynucleotide sequence to beamplified is flanked by primer hybridization sites, such as ahybridization site for a 5′ primer (or the complement thereof) and ahybridization site for a 3′ primer (or the complement thereof). Atemplate may comprise RNA and/or DNA, and may be from a natural source,or be synthetic. Nonlimiting exemplary templates include genomic DNA,viral DNA, mitochondrial DNA, viral RNA, mRNA, tRNA, microRNA, plasmids,vectors, cosmids, artificial chromosomes, etc. Any polynucleotide thatmay be copied or amplified by a polymerase enzyme is considered atemplate.

“Domain” refers to a unit of a protein or protein complex, comprising apolypeptide subsequence, a complete polypeptide sequence, or a pluralityof polypeptide sequences where that unit has a defined function. Thefunction is understood to be broadly defined and can be ligand binding,catalytic activity, and/or can have a stabilizing effect on thestructure of the protein.

Residues “correspond” to each other where they occur at equivalentpositions in aligned amino acid sequences, such as family B thermophilicpolymerase sequences and/or a domain thereof, such as a catalytic orexonuclease domain. Corresponding positions can be identified aspositions that align with one another. Related or variant polypeptidesare aligned by any method in the art. Such methods typically maximizematches, and include methods such as using manual alignments and byusing any of the numerous alignment programs available (for example,BLASTP) and others known in the art. By aligning the sequences ofpolypeptides, one of skill in the art can identify correspondingresidues, using conserved and identical amino acid residues as guides.In some embodiments, an amino acid of a polypeptide is considered tocorrespond to an amino acid in a disclosed sequence when the amino acidof the polypeptide is aligned with the amino acid in the disclosedsequence upon alignment of the polypeptide with the disclosed sequenceto maximize identity and homology (e.g., where conserved amino acids arealigned) using a standard alignment algorithm, such as the BLASTPalgorithm with default scoring parameters (such as, for example,BLOSUM62 Matrix, Gap existence penalty 11, Gap extension penalty 1, andwith default general parameters). As a non-limiting example, withreference to the multiple sequence alignment shown in FIGS. 11A-C, aminoacid residue 408 in SEQ ID NO: 9 corresponds to positions 410, 407, 407,407, and 408 in SEQ ID NOs: 52, 57, 55, 56, and 51, respectively (markedwith an asterisk in FIG. 11A). As another non-limiting example, aminoacid residue 762 in SEQ ID NO: 9 corresponds to positions 764, 761, 761,761, and 762 in SEQ ID NOs: 52, 57, 55, 56, and 51, respectively (markedwith a pound in FIG. 11B). In some embodiments, corresponding positionscan also be identified using overlaid 3-D structures, where available,as positions at which greater than 50% of the volume occupied by aspace-filling model of an amino acid in a first polypeptide is occupiedby the space-filling model of the corresponding amino acid in a secondpolypeptide.

“Identity” is measured by a score determined by comparing the amino acidsequences of the two polypeptides using the Bestfit program. Bestfituses the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981) to find the best segment ofsimilarity between two sequences. When using Bestfit to determinewhether a test amino acid sequence is, for instance, 95% identical to areference sequence according to the present disclosure, the parametersare set so that the percentage of identity is calculated over the fulllength of the test amino acid sequence, such that 95% of the amino acidsin the test amino acid sequence align with identical amino acids on thereference sequence.

“Sequence-non-specific DNA binding domain” or “DNA binding domain”refers to a protein domain that binds to DNA without significantsequence preference. In some embodiments, a DNA binding domain binds todouble-stranded DNA. Non-limiting exemplary DNA binding domains includeSso7d from Sulfolobus solfataricus, Sac7d, Sac7a, Sac7b, and Sac7e fromS. acidocaldarius, and Ssh7a and Ssh7b from Sulfolobus shibatae,Pae3192, Pae0384, and Ape3192, HMf family archaeal histone domains, andarchaeal PCNA homolog.

With reference to two polypeptides or two polypeptide domains, the term“fused” means that the two polypeptides or polypeptide domains arecontained in a single contiguous polypeptide sequence.

“Heterologous”, when used with reference to portions of a protein,indicates that the protein comprises two or more domains that are notfound in the same relationship to each other in nature. In someembodiments, such a protein, e.g., a fusion protein, contains two ormore domains from unrelated proteins arranged to make a new functionalprotein.

“Error-correcting activity” of a polymerase or polymerase domain refersto the 3′ to 5′ exonuclease proofreading activity of a polymerasewhereby nucleotides that do not form Watson-Crick base pairs with thetemplate are removed from the 3′ end of an oligonucleotide, i.e., astrand being synthesized from a template, in a sequential manner.Examples of polymerases that have error-correcting activity includepolymerases from Pyrococcus furiosus, Thermococcus litoralis, andThermotoga maritima with wild-type exonuclease domains, and certainothers discussed herein.

“Sensitivity” as used herein, refers to the ability of a polymerase toamplify a target nucleic acid that is present at low copy number. Insome embodiments, low copy number refers to a target nucleic acid thatis present at fewer than 10,000 or fewer than 1,000 or fewer than 100 orfewer than 10 copies in the composition comprising the target nucleicacid and the polymerase.

“Specificity” as used herein, refers to the ability of a polymerase toamplify a target nucleic acid while producing fewer non-specificamplification byproducts, such as those resulting from primer-dimers.

As used herein the terms “hybridize”, “hybridizing”, “hybridization” andother related terms include hydrogen bonding between two differentnucleic acids, or between two different regions of a nucleic acid, toform a duplex nucleic acid. Hybridization can comprise Watson-Crick orHoogstein binding to form a duplex nucleic acid. The two differentnucleic acids, or the two different regions of a nucleic acid, may becomplementary, or partially complementary. The complementary basepairing can be the standard A-T or C-G base pairing, or can be otherforms of base-pairing interactions. Duplex nucleic acids can includemismatched base-paired nucleotides. Complementary nucleic acid strandsneed not hybridize with each other across their entire length.

In some embodiments, conditions that are suitable for nucleic acidhybridization and/or nucleic acid synthesis include parameters such assalts, buffers, pH, temperature, % GC content of the polynucleotide andprimers, and/or time. For example, conditions suitable for hybridizingnucleic acids (e.g., polynucleotides and primers) can includehybridization solutions having sodium salts, such as NaCl, sodiumcitrate and/or sodium phosphate. In some embodiments, a hybridizationsolution can be a stringent hybridization solution which can include anycombination of formamide (e.g., about 50%), 5×SSC (e.g., about 0.75 MNaCl and about 0.075 M sodium citrate), sodium phosphate (e.g., about 50mM at about pH 6.8), sodium pyrophosphate (e.g., about 0.1%),5×Denhardt's solution, SDS (e.g., about 0.1%), and/or dextran sulfate(e.g., about 10%). In some embodiments, hybridization and/or nucleicacid synthesis can be conducted at a temperature range of about 45-55°C., or about 55-65° C., or about 65-75° C.

In some embodiments, hybridization or nucleic acid synthesis conditionscan be conducted at a pH range of about 5-10, or about pH 6-9, or aboutpH 6.5-8, or about pH 6.5-7.

Thermal melting temperature (T_(m)) for nucleic acids can be atemperature at which half of the nucleic acid strands aredouble-stranded and half are single-stranded under a defined condition.In some embodiments, a defined condition can include ionic strength andpH in an aqueous reaction condition. A defined condition can bemodulated by altering the concentration of salts (e.g., sodium),temperature, pH, buffers, and/or formamide. Typically, the calculatedthermal melting temperature can be at about 5-30° C. below the T_(m), orabout 5-25° C. below the T_(m), or about 5-20° C. below the T_(m), orabout 5-15° C. below the T_(m), or about 5-10° C. below the T_(m).Methods for calculating a T_(m) are well known and can be found inSambrook (1989 in “Molecular Cloning: A Laboratory Manual”, 2^(nd)edition, volumes 1-3; Wetmur 1966, J. Mol. Biol., 31:349-370; Wetmur1991 Critical Reviews in Biochemistry and Molecular Biology,26:227-259). Other sources for calculating a T_(m) for hybridizing ordenaturing nucleic acids include OligoAnalyze (from Integrated DNATechnologies) and Primer3 (distributed by the Whitehead Institute forBiomedical Research).

Provided herein are thermophilic DNA polymerases comprising a family Bpolymerase catalytic domain in which a neutral amino acid residue ispresent at a certain position. Many types of Family B polymerases aredescribed in Rothwell and Watsman, Advances in Protein Chemistry71:401-440 (2005). Examples of thermophilic Family B polymerases includethose of the Pyrococcus and Thermococcus genera, such as the Deep Ventpolymerase and Family B polymerases of P. furiosus, P. calidifontis, P.aerophilum, T kodakarensis, T gorgonarius, and Thermococcus sp. 9° N-7.Exemplary wild-type amino acid sequences for such thermophilic family Bpolymerases can be obtained from public databases such as NCBI GenBankor UniProt. Wild-type sequences include naturally-occurring variants ofthe amino acid sequences for such thermophilic family B polymerases canbe obtained from public databases such as NCBI GenBank or UniProt. Notethat in some cases, the sequences are annotated as containing inteins;the inteins are not present in the mature enzyme.

In some embodiments, the family B polymerase catalytic domain has anamino acid sequence in which the position corresponding to position 379of SEQ ID NO: 6 is a neutral amino acid residue. In some embodiments,the family B polymerase catalytic domain has an amino acid sequencewherein the amino acid residue at the position of the amino acidsequence that aligns to position 379 of SEQ ID NO: 6 is a neutral aminoacid residue. In some embodiments, the family B polymerase catalyticdomain has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to asequence selected from SEQ ID NOs: 6 to 10, 15 to 18, 25, 26, 33, 34,37, 38, 41, 42, and 45 to 48, wherein the position corresponding toposition 379 of SEQ ID NO: 6 is a neutral amino acid residue. In someembodiments, the family B polymerase catalytic domain has at least 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 7, wherein theposition corresponding to position 379 of SEQ ID NO: 6 is a neutralamino acid residue. In some embodiments, the family B polymerasecatalytic domain has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%identity to the catalytic domain of SEQ ID NO: 15, wherein the positioncorresponding to position 379 of SEQ ID NO: 6 is a neutral amino acidresidue. In some embodiments, the family B polymerase catalytic domainhas at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to thecatalytic domain of SEQ ID NO: 25, wherein the position corresponding toposition 379 of SEQ ID NO: 6 is a neutral amino acid residue. In someembodiments, the family B polymerase catalytic domain has at least 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the catalytic domain of SEQID NO: 33, wherein the position corresponding to position 379 of SEQ IDNO: 6 is a neutral amino acid residue. In some embodiments, the family Bpolymerase catalytic domain has at least 80%, 85%, 90%, 95%, 98%, 99%,or 100% identity to the catalytic domain of SEQ ID NO: 37, wherein theposition corresponding to position 379 of SEQ ID NO: 6 is a neutralamino acid residue. In some embodiments, the family B polymerasecatalytic domain has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%identity to the catalytic domain of SEQ ID NO: 47, wherein the positioncorresponding to position 379 of SEQ ID NO: 6 is a neutral amino acidresidue. In some embodiments, the family B polymerase catalytic domainhas at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to thecatalytic domain of SEQ ID NO: 41, wherein the position corresponding toposition 379 of SEQ ID NO: 6 is a neutral amino acid residue. In someembodiments, the family B polymerase catalytic domain has at least 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the catalytic domain of SEQID NO: 45, wherein the position corresponding to position 379 of SEQ IDNO: 6 is a neutral amino acid residue.

Examples of family B polymerase catalytic domain sequences are shown,e.g., in FIG. 12 . In some embodiments, the C-terminus is the residue atthe position of the conserved leucine shown as the last residue in themultiple sequence alignment in FIG. 12 . In some embodiments, theC-terminus of the family B polymerase catalytic domain is the positioncorresponding to position 383 of SEQ ID NO: 6. In some embodiments, theC-terminus of the family B polymerase catalytic domain is the positioncorresponding to the leucine which is the last residue of SEQ ID NO: 6.In some embodiments, the C-terminus of the family B polymerase catalyticdomain is the position that aligns to the leucine which is the lastresidue of SEQ ID NO: 6. In some embodiments, the C-terminus of thefamily B polymerase catalytic domain is the position corresponding to aleucine selected from the leucines shown as the final residues in FIG.12 . In some embodiments, the C-terminus of the family B polymerasecatalytic domain is the position that aligns to a leucine selected fromthe leucines shown as the final residues in FIG. 12 . The C-terminalresidue in any of the foregoing embodiments can be a leucine.

In some embodiments, the thermophilic DNA polymerase comprises: (a) theconsecutive amino acid residues WQKTX, (b) the consecutive amino acidresidues YQKTX, (c) the consecutive amino acid residues XQTGL, (d) theconsecutive amino acid residues XQVGL, (e) the consecutive amino acidresidues KTXQT, or (f) the consecutive amino acid residues KTXQV;wherein X is a neutral amino acid residue; and wherein X is within 20,15, 10, 5, or 4 residues of the C-terminus of the family B polymerasecatalytic domain. In some embodiments, The C-terminus of the family Bpolymerase catalytic domain can be identified as the amino acid thataligns to or corresponds to the last amino acid of SEQ ID NO: 6. In someembodiments, the thermophilic DNA polymerase comprises a consecutiveamino acid sequence of WQKTX, XQTGL, KTXQT, YQKTX, XQVGL, KTXQV, YQSSX,XQTGL, or SSXQT; wherein X is a neutral amino acid residue; and whereinX is within 20, 15, 10, 5, or 4 residues of the C-terminus of the familyB polymerase catalytic domain. In some embodiments, the thermophilic DNApolymerase comprises a consecutive amino acid sequence of WQKTX, XQTGL,KTXQT, YQKTX, XQVGL, KTXQV, YQSSX, XQTGL, SSXQT, TGRVX, XKSLL, RVXKS,TGRSX, XRTLL, or RSXRT; wherein Xis a neutral amino acid residue; andwherein Xis within 20, 15, 10, 5, or 4 residues of the C-terminus of thefamily B polymerase catalytic domain. X can be within 15 residues of theC-terminus of the family B polymerase catalytic domain in any of theforegoing embodiments. X can be within 10 residues of the C-terminus ofthe family B polymerase catalytic domain in any of the foregoingembodiments. X can be within 5 residues of the C-terminus of the familyB polymerase catalytic domain in any of the foregoing embodiments. X canbe within 4 residues of the C-terminus of the family B polymerasecatalytic domain in any of the foregoing embodiments. For the avoidanceof doubt, in a sequence segment consisting of n residues, residues 1 ton are within n−1 residues of position n; e.g., if n is 5, positions 1,2, 3, 4, and 5 and are within 4 residues of position 5.

This paragraph concerns the neutral amino acid residue referred to inany of the embodiments mentioned in the preceding paragraphs. Neutralamino acid residues do not have side chains containing groups that aremore than 50% charged at pH 7.4 in aqueous solution at 37° C., such ascarboxyls, amines, and guanidino groups. Neutral amino acid residuesinclude canonical and noncanonical residues unless indicated to thecontrary. In some embodiments, the neutral amino acid is a noncanonicalresidue. A noncanonical residue is a residue other than the twenty aminoacid residues abbreviated as one of the twenty following letters: A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y (e.g., norleucineand selenomethionine are noncanonical; see, e.g., U.S. Pat. No.7,541,170 for additional examples of noncanonical residues, which arereferred to therein as “nonclassical amino acids or chemical amino acidanalogs”). In some embodiments, the neutral amino acid is less than 10%,1%, 0.1%, or 0.01% charged at pH 7.4 in aqueous solution at 37° C. Insome embodiments, the neutral amino acid residue is a polar neutralamino acid residue. A residue is polar if its side chain contains atleast one hydrogen bond donor or acceptor. In some embodiments, theneutral amino acid comprises a side chain comprising an alcohol, amide,carbonyl, ester, or ether. In some embodiments, the neutral amino acidcomprises a side chain comprising an alcohol. In some embodiments, theneutral amino acid comprises a side chain comprising an amide. In someembodiments, the neutral amino acid residue is Q, N, H, S, T, Y, C, M,W, A, I, L, F, V, P, or G. In some embodiments, the neutral amino acidresidue is Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, or G. In someembodiments, the neutral amino acid residue is Q, N, S, T, C, M, A, I,L, V, or G. In some embodiments, the neutral amino acid residue is Q, N,S, T, C, M, A, or G. In some embodiments, the neutral amino acid residueis Q, N, H, S, T, Y, C, M, or W. In some embodiments, the neutral aminoacid residue is Q, N, H, S, T, Y, or W. In some embodiments, the neutralamino acid residue is Q, N, H, S, T, C, or M. In some embodiments, theneutral amino acid residue is Q, N, S, T, C, or M. In some embodiments,the neutral amino acid residue is Q, N, S, or T. In some embodiments,the neutral amino acid residue is Q or N. In some embodiments, theneutral amino acid residue is S. In some embodiments, the neutral aminoacid residue is T. In some embodiments, the neutral amino acid residueis Q. In some embodiments, the neutral amino acid residue is N.

In some embodiments, the family B polymerase catalytic domain is asubfamily B3 polymerase domain. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain of aPyrococcus in which a neutral amino acid residue is present at aposition discussed above. In some embodiments, the family B polymerasecatalytic domain is a family B polymerase domain of a Thermococcus inwhich a neutral amino acid residue is present at a position discussedabove. In some embodiments, the family B polymerase catalytic domain isa family B polymerase domain of a Pyrobaculum in which a neutral aminoacid residue is present at a position discussed above. In someembodiments, the family B polymerase catalytic domain is a family Bpolymerase domain of Pyrococcus furiosus in which a neutral amino acidresidue is present at a position discussed above. In some embodiments,the family B polymerase catalytic domain is a family B polymerase domainof Pyrococcus species GB-D in which a neutral amino acid residue ispresent at a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofThermococcus kodakarensis in which a neutral amino acid residue ispresent at a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofThermococcus litoralis in which a neutral amino acid residue is presentat a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofThermococcus gorgonarius in which a neutral amino acid residue ispresent at a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofThermococcus sp. 9° N-7 in which a neutral amino acid residue is presentat a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofPyrobaculum calidifontis in which a neutral amino acid residue ispresent at a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofPyrobaculum aerophilum in which a neutral amino acid residue is presentat a position discussed above.

In some embodiments, all domains of the thermophilic DNA polymerase arecontained in a single polypeptide. In some embodiments, the thermophilicDNA polymerase comprises a plurality of polypeptide chains, which may benoncovalently associated or covalently associated. The plurality ofpolypeptide chains can include a first polypeptide comprising apolymerase catalytic domain and a second polypeptide comprising anadditional domain, such as a sequence non-specific double-strandedDNA-binding domain. A covalent association can include, e.g., one ormore disulfide bonds or chemical conjugation using a linking compound,e.g., a chemical crosslinking agent, including, for example,succinimidyl-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC).Disulfide bonds and chemical conjugation are discussed further below.

In some embodiments, the thermophilic DNA polymerase comprises asequence non-specific DNA-binding domain, e.g., a thermostable DNAbinding domain. The DNA binding domain can be, for example, present aspart of a fusion protein with the polymerase catalytic domain. In someembodiments, the DNA binding domain is fused C-terminal to thepolymerase catalytic domain. In some embodiments, the DNA binding domainis noncovalently associated with the polypeptide comprising thepolymerase catalytic domain, e.g., in the manner of the associationbetween sliding clamps and certain family B polymerases. In someembodiments, the polypeptide comprising the polymerase catalytic domainfurther comprises a sequence that noncovalently associates with an DNAbinding domain, such as the PCNA-interacting sequence of a dimericarchaeal polymerase such as Pfu Pol II. As discussed, e.g., in U.S. Pat.No. 7,541,170, an DNA binding domain can provide improved processivityrelative to version of the enzyme lacking the DNA binding domain.Processivity reflects the extent to which a polymerase continues tosynthesize DNA (adding nucleotides in processive catalytic events) alongthe same template without falling off. In some embodiments, highprocessivity correlates to high sensitivity in amplification reactions.

In some embodiments, the DNA binding domain is covalently conjugated tothe polypeptide comprising the polymerase catalytic domain. Techniquesfor covalent conjugation of heterologous domains are described, e.g., inBIOCONJUGATE TECHNIQUES, Hermanson, Ed., Academic Press (1996). Suchtechniques include, for example, derivitization for the purpose oflinking the moieties to each other, either directly or through a linkingcompound, by methods that are well known in the art of proteinchemistry. For example, in one chemical conjugation embodiment, thecatalytic domain and the nucleic acid binding domain are linked using aheterobifunctional coupling reagent which ultimately contributes toformation of an intermolecular disulfide bond between the two moieties.Other types of coupling reagents that are useful in this capacity forthe present invention are described, for example, in U.S. Pat. No.4,545,985. Alternatively, an intermolecular disulfide may convenientlybe formed between cysteines in each moiety, which occur naturally or areinserted by genetic engineering. The means of linking moieties may alsouse thioether linkages between heterobifunctional crosslinking reagentsor specific low pH cleavable crosslinkers or specific protease cleavablelinkers or other cleavable or noncleavable chemical linkages.

In some embodiments, the sequence non-specific double-strandedDNA-binding domain comprises an amino acid sequence having at least 75%,80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to an amino acid sequenceselected from SEQ ID NOs: 53 to 62. In some embodiments, the DNA bindingdomain is an archaeal DNA binding domain. In some embodiments, the DNAbinding domain is a 7 kD DNA-binding domain, which occurs in certainarchaeal small basic DNA binding proteins (see, e.g., Choli et al.,Biochimica et Biophysica Acta 950:193-203, 1988; Baumann et al.,Structural Biol. 1:808-819, 1994; and Gao et al, Nature Struc. Biol.5:782-786, 1998). Additional archaeal DNA binding domains are discussedin Hardy and Martin, Extremophiles 12:235-46 (2008).

In some embodiments, the DNA binding domain is an Sso7d domain. In someembodiments, the DNA binding domain is a Sac7d domain. In someembodiments, the DNA binding domain is a Sac7e domain. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 53. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 54. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 62.

In some embodiments, the DNA binding domain is a Pae3192 domain. In someembodiments, the DNA binding domain is a Pae0384 domain. In someembodiments, the DNA binding domain is a Ape3192 domain. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 55. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 56. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 57.

In some embodiments, the DNA binding domain is an archaeal histonedomain. In some embodiments, the archaeal histone domain is an HIVIffamily archaeal histone domain (see, e.g., Starich et al., J Molec.Biol. 255:187-203, 1996; Sandman et al., Gene 150:207-208, 1994). Insome embodiments, the archaeal histone domain is an HIVIf familyarchaeal histone domain from Methanothermus. In some embodiments, thearchaeal histone domain is an HIVIf family archaeal histone domain fromPyrococcus. In some embodiments, the archaeal histone domain is an HMffamily archaeal histone domain from Methanothermus fervidus. In someembodiments, the archaeal histone domain is an HIVIf family archaealhistone domain from Pyrococcus strain GB-3a. In some embodiments, thearchaeal histone domain is a Methanothermus HMfA archaeal histonedomain. In some embodiments, the archaeal histone domain is aMethanothermus HMfB archaeal histone domain. In some embodiments, thearchaeal histone domain is a Pyrococcus HpyA1 archaeal histone domain.In some embodiments, the archaeal histone domain is a Pyrococcus HpyA2archaeal histone domain. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 58. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 59. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 60. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 61.

In some embodiments, the DNA binding domain is a sliding clamp, such asan archaeal PCNA homolog. Sliding clamps can exist as trimers insolution, and can form a ring-like structure with a central passagecapable of accommodating double-stranded DNA. The sliding clamp formsspecific interactions with the amino acids located at the C terminus ofparticular DNA polymerases, and tethers those polymerases to the DNAtemplate during replication. The sliding clamp in eukaryotes is referredto as the proliferating cell nuclear antigen (PCNA), while similarproteins in other domains are often referred to as PCNA homologs. Thesehomologs have marked structural similarity but limited sequencesimilarity. PCNA homologs have been identified from thermophilic Archaea(e.g., Sulfolobus solfataricus, Pyrococcus furiosus, etc.). Some familyB polymerases in Archaea have a C terminus containing a consensusPCNA-interacting amino acid sequence and are capable of using a PCNAhomolog as a processivity factor (see, e.g., Cann et al., J. Bacteriol.181:6591-6599, 1999 and De Felice et al., J Mol. Biol. 291:47-57, 1999).These PCNA homologs are useful sequence-non-specific double-stranded DNAbinding domains. For example, a consensus PCNA-interacting sequence canbe joined to a polymerase that does not naturally interact with a PCNAhomolog, thereby allowing a PCNA homolog to serve as a processivityfactor for the polymerase. By way of illustration, the PCNA-interactingsequence from Pyrococcus furiosus Pol II (a heterodimeric DNA polymerasecontaining two family B-like polypeptides) can be covalently joined to asequence based on Pyrococcus furiosus Pol I (a monomeric family Bpolymerase that does not normally interact with a PCNA homolog). Theresulting fusion protein can then be allowed to associate non-covalentlywith the Pyrococcus furiosus PCNA homolog to generate a heterologousprotein with increased processivity.

Nucleic acids encoding the domains of a fusion protein invention can beobtained using recombinant genetics techniques. Basic texts disclosingthe general methods for doing so include Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL (2nd ed. 1989); Kriegler, GENE TRANSFER ANDEXPRESSION:A LABORATORY MANUAL (1990); and CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Ausubel et al., eds., 1994)).

In some embodiments, catalytic and binding domains of the polymerase arejoined by a linker domain, e.g., a polypeptide sequence of 1 to about200 amino acids in length, such as 1 to about 100, 50, 25, or 10 aminoacids. In some embodiments, proline residues are incorporated into thelinker to prevent the formation of significant secondary structuralelements by the linker. Linkers can often be flexible amino acidsubsequences that are synthesized as part of a recombinant fusionprotein. For a discussion of linkers, see, e.g., US 2011/0086406 A1including at paragraphs 83-89 thereof.

In some embodiments, the thermophilic DNA polymerase comprises anexonuclease domain. In some embodiments, the exonuclease domain is a 3′to 5′ exonuclease domain. The 3′-5′ exonuclease domain can haveerror-correcting activity, also known as proofreading activity, in whichthe exonuclease preferentially removes a base from a nascent DNAstrand/extension product/3′ terminus that is not a Watson-Crick match tothe template strand. In some embodiments, the 3′-5′ exonuclease domainis a DEDDy archaeal exonuclease domain. In some embodiments, theexonuclease domain is N-terminal to the DNA polymerase catalytic domain.In some embodiments, the thermophilic DNA polymerase comprises a domainhaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 63. In some embodiments, the thermophilic DNA polymerasecomprises a domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% identity to the exonuclease domain of a sequence selected from SEQID NO: 1, 19, 23, 31, 35, 39, 43, 49, 51, or 52. In some embodiments,the thermophilic DNA polymerase comprises a domain having at least 75%,80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the exonuclease domainof SEQ ID NO: 1. In some embodiments, the thermophilic DNA polymerasecomprises a domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% identity to the exonuclease domain of SEQ ID NO: 19. In someembodiments, the thermophilic DNA polymerase comprises a domain havingat least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to theexonuclease domain of SEQ ID NO: 23. In some embodiments, thethermophilic DNA polymerase comprises a domain having at least 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the exonuclease domain ofSEQ ID NO: 31. In some embodiments, the thermophilic DNA polymerasecomprises a domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% identity to the exonuclease domain of SEQ ID NO: 35. In someembodiments, the thermophilic DNA polymerase comprises a domain havingat least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to theexonuclease domain of SEQ ID NO: 39. In some embodiments, thethermophilic DNA polymerase comprises a domain having at least 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the exonuclease domain ofSEQ ID NO: 43. In some embodiments, the thermophilic DNA polymerasecomprises a domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% identity to the exonuclease domain of SEQ ID NO: 49. In someembodiments, the thermophilic DNA polymerase comprises a domain havingat least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to theexonuclease domain of SEQ ID NO: 51. In some embodiments, thethermophilic DNA polymerase comprises a domain having at least 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the exonuclease domain ofSEQ ID NO: 52. An exonuclease domain can be identified using BLASTPagainst the RefSeq database can be identified by using NCBI BLASTP tosearch the RefSeq database. NCBI BLASTP automatically identifies certaindomains such as exonuclease domains and indicates their termini as thepositions at which the domain begins and ends.

In some embodiments, the exonuclease domain is an exonuclease domain ofa Pyrococcus. In some embodiments, the exonuclease domain is anexonuclease domain of a Thermococcus. In some embodiments, theexonuclease domain is an exonuclease domain of a Pyrobaculum. In someembodiments, the exonuclease domain is an exonuclease domain ofPyrococcus furiosus. In some embodiments, the exonuclease domain is anexonuclease domain of Pyrococcus species GB-D. In some embodiments, theexonuclease domain is an exonuclease domain of Thermococcuskodakarensis. In some embodiments, the exonuclease domain is anexonuclease domain of Thermococcus litoralis. In some embodiments, theexonuclease domain is an exonuclease domain of Thermococcus gorgonarius.In some embodiments, the exonuclease domain is an exonuclease domain ofThermococcus sp. 9° N-7. In some embodiments, the exonuclease domain isan exonuclease domain of Pyrobaculum calidifontis. In some embodiments,the exonuclease domain is an exonuclease domain of Pyrobaculumaerophilum.

In some embodiments, the thermophilic DNA polymerase comprises aninactivated or reduced-activity exonuclease domain. An inactivatedexonuclease domain is a mutated version of a wild-type domain that hasless than 50% of the wild-type exonuclease activity. In someembodiments, the inactivated domain has less than 40%, less than 30%,less than 25%, less than 20%, less than 15%, less than 10%, or less than5% of the wild-type exonuclease activity. In some embodiments, theinactivated domain has less than 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%,or 0.01% of the wild-type exonuclease activity. A reduced-activityexonuclease domain is a mutated version of a wild-type domain that hasless than 10% of the wild-type exonuclease activity. Measurement ofexonuclease activity is described, for example, in DNA Replication 2nd,edition, by Kornberg and Baker, W.H. Freeman & Company, New York, N.Y.1991. Examples of exo⁻ DNA polymerase mutants include those with asingle mutation in Motif I and/or II (Motifs are as described, e.g., inU.S. Pat. No. 8,921,043, e.g., at FIG. 2 ), or a double mutation inMotif I (such as D141A and E143A, the position numbering corresponds toPfu polymerase, SEQ ID NO: 1), that reportedly abolishes detectibleexonuclease activity (see for example, VENT® (Thermococcus litoralis)(Kong et al. J. Biol. Chem. 268(3):1965-1975) (New England Biolabs, Inc.(NEB), Ipswich, Mass.); Thermococcus JDF-3 (U.S. Pat. No. 6,946,273,U.S. 2005/0069908); KODI (Thermococcus kodakaraensis) (U.S. Pat. No.6,008,025); Pfu (Pyrococcus furiosus) (U.S. Pat. Nos. 5,489,523,7,704,712, and 7,659,100); and 9° N (Thermococcus sp.) (U.S.2005/0123940 and Southworth et al. Proc Natl Acad Sci USA 93:5281-5285(1996)); see also U.S. Pat. No. 8,921,043. In some embodiments, theexonuclease domain has a D141A, E143A, D215A, D315A, D141A/E143A,D141A/D315A, E143A/D315A, D215A/D315A, or D141A/E143A/D315A mutation. Insome embodiments, the exonuclease domain has an A, N, S, T, or E residueat the position corresponding to position 141 of SEQ ID NO: 1. In someembodiments, the exonuclease domain has an A at the positioncorresponding to position 141 of SEQ ID NO: 1. In some embodiments, theexonuclease domain has an A at the position corresponding to position143 of SEQ ID NO: 1.

In some embodiments, the amino acid residue at the position of thefamily B polymerase catalytic domain amino acid sequence that aligns toposition 25 of SEQ ID NO: 6 is a serine. In some embodiments, the aminoacid residue at the position of the family B polymerase catalytic domainamino acid sequence that corresponds to position 25 of SEQ ID NO: 6 is aserine. In some embodiments, the thermophilic DNA polymerase comprises:(a) the consecutive amino acid residues LDFRS, (b) the consecutive aminoacid residues FRSLY, or (c) the consecutive amino acid residues SLYPS,wherein the underlined serine residue is within 30 amino acid residuesof the N-terminus of the family B polymerase catalytic domain. In someembodiments, the thermophilic DNA polymerase comprises:

(a) the consecutive amino acid residues LDFRS*, (b) the consecutiveamino acid residues FRS*LY, or (c) the consecutive amino acid residuesS*LYPS, wherein the serine residue immediately followed by an asteriskis within 30 amino acid residues of the N-terminus of the family Bpolymerase catalytic domain. The asterisk is included solely todesignate the serine that is within 30 amino acid residues of theN-terminus of the family B polymerase catalytic domain and does notsignify a structural difference. In some embodiments, the N-terminus ofthe family B polymerase catalytic domain is the residue immediatelypreceding the conserved tyrosine shown as the second residue in themultiple sequence alignment in FIG. 12 . In some embodiments, theN-terminus of the family B polymerase catalytic domain is the positionimmediately preceding the position corresponding to the first tyrosinein SEQ ID NO: 6. In some embodiments, the N-terminus of the family Bpolymerase catalytic domain is the position that aligns to position 1 ofSEQ ID NO: 6. In some embodiments, the N-terminus of the family Bpolymerase catalytic domain is the position corresponding to theposition immediately preceding a tyrosine selected from the tyrosinesshown as the second residues in FIG. 12 . In some embodiments, theN-terminus of the family B polymerase catalytic domain is the positionimmediately preceding the position that aligns to a tyrosine selectedfrom the tyrosines shown as the second residues in FIG. 12 . In someembodiments, the N-terminus of the family B polymerase catalytic domainis the position immediately preceding the position corresponding to atyrosine selected from the tyrosines shown as the second residues inFIG. 12 . The N-terminal residue in any of the foregoing embodiments canbe a serine. The N-terminal residue in any of the foregoing embodimentscan be a threonine. The N-terminal residue in any of the foregoingembodiments can be a glycine. The N-terminal residue in any of theforegoing embodiments can be a proline.

As will be apparent from various aspects of the discussion above, familyB polymerases are well-characterized in general and are known totolerate mutations at a number of positions. Furthermore, the followingis a non-exhaustive list of patents and published applications thatdiscuss mutations in family B polymerases and the properties of mutatedfamily B polymerases: U.S. Pat. Nos. 8,435,775; 8,557,554;WO2007/016702; US 2003/0180741; WO 2004/011605; WO 2003/060144; and U.S.Pat. No. 9,023,633. Accordingly, those skilled in the art willunderstand in view of this disclosure that the residues discussed hereinsuch as the neutral amino acid at the position corresponding to position379 of SEQ ID NO: 6 can be incorporated into a wide variety ofthermophilic family B polymerases and can be accompanied by other aminoacid residues that differ from wild-type residues. Thus, in someembodiments, the thermophilic DNA polymerase comprises an amino acidsequence comprising at least one difference from SEQ ID NO: 1 at aposition corresponding to position 15, 72, 93, 141, 143, 247, 265, 337,385, 387, 388, 399, 400, 405, 407, 410, 485, 542, 546, 593, or 595 ofSEQ ID NO: 1. In some embodiments, the thermophilic DNA polymerasecomprises an amino acid sequence comprising at least one missing residuecorresponding to position 92, 93, 94, or 381 of SEQ ID NO: 1. In someembodiments, the at least one difference or missing residue is in theexonuclease domain. In some embodiments, the at least one difference ormissing residue is in the polymerase catalytic domain.

In some embodiments, the polymerase with the at least one difference ormissing residue has an expanded substrate range relative to a polymerasewithout the difference or in which the residue is not missing. In someembodiments, the at least one difference comprises a G or D at theposition corresponding to position 400 of SEQ ID NO: 1. In someembodiments, the at least one difference comprises an I at the positioncorresponding to position 407 of SEQ ID NO: 1. In some embodiments, theat least one difference comprises an I at the position corresponding toposition 337 of SEQ ID NO: 1. In some embodiments, the at least onedifference comprises a D at the position corresponding to position 399of SEQ ID NO: 1. In some embodiments, the at least one differencecomprises an H at the position corresponding to position 546 of SEQ IDNO: 91.

In some embodiments, the polymerase with the at least one difference ormissing residue incorporates a nucleotide analog to a greater extentthan a polymerase without the difference or in which the residue is notmissing. In some embodiments, the at least one difference comprises an Lat the position corresponding to position 410 of SEQ ID NO: 1. In someembodiments, the at least one difference comprises a T at the positioncorresponding to position 485 of SEQ ID NO: 1.

In some embodiments, the polymerase with the at least one difference ormissing residue has reduced uracil sensitivity relative to a polymerasewithout the difference or in which the residue is not missing. In someembodiments, the at least one missing residue comprises a missingresidue at the position corresponding to position 93 of SEQ ID NO: 1. Insome embodiments, the at least one missing residue comprises a missingresidue at the position corresponding to position 94 of SEQ ID NO: 1. Insome embodiments, the at least one missing residue comprises a missingresidue at the position corresponding to position 92 of SEQ ID NO: 1. Insome embodiments, the at least one difference comprises a Q, R, E, A, K,N, or G at the position corresponding to position 93 of SEQ ID NO: 1. Insome embodiments, the at least one difference comprises a Q or R at theposition corresponding to position 93 of SEQ ID NO: 1. In someembodiments, an at least one difference or missing residue as discussedabove in this paragraph is accompanied by at least one difference ormissing residue that offsets a loss of activity. In some embodiments,the at least one difference that offsets a loss of activity comprises anR at the position corresponding to position 247 of SEQ ID NO: 1. In someembodiments, the at least one difference that offsets a loss of activitycomprises an R at the position corresponding to position 265 of SEQ IDNO: 1. In some embodiments, the at least one difference that offsets aloss of activity comprises an R at the position corresponding toposition 485 of SEQ ID NO: 1. In some embodiments, the at least onemissing residue that offsets a loss of activity comprises a missingresidue at the position corresponding to position 381 of SEQ ID NO: 1.

In some embodiments, the at least one difference comprises an R at theposition corresponding to position 247 of SEQ ID NO: 1. In someembodiments, the at least one difference comprises an R at the positioncorresponding to position 265 of SEQ ID NO: 1. In some embodiments, theat least one difference comprises an R at the position corresponding toposition 485 of SEQ ID NO: 1. In some embodiments, the at least onemissing residue comprises a missing residue at the positioncorresponding to position 381 of SEQ ID NO: 1. In some embodiments, theat least one difference comprises an I at the position corresponding toposition 15 of SEQ ID NO: 1. In some embodiments, the at least onedifference comprises an R at the position corresponding to position 72of SEQ ID NO: 1.

In some embodiments, the polymerase with the at least one difference ormissing residue has an altered proofreading spectrum relative to apolymerase without the difference or in which the residue is notmissing. In some embodiments, the at least one difference comprises a Por S at the position corresponding to position 387 of SEQ ID NO: 1. Insome embodiments, the at least one difference comprises an E at theposition corresponding to position 405 of SEQ ID NO: 1. In someembodiments, the at least one difference comprises an F at the positioncorresponding to position 410 of SEQ ID NO: 1. In some embodiments, theat least one difference comprises a P at the position corresponding toposition 542 of SEQ ID NO: 1. In some embodiments, the at least onedifference comprises a T at the position corresponding to position 593of SEQ ID NO: 1. In some embodiments, the at least one differencecomprises an S at the position corresponding to position 595 of SEQ IDNO: 1. In some embodiments, the at least one difference comprises a Q,S, N, L, or H at the position corresponding to position 385 of SEQ IDNO: 1. In some embodiments, the at least one difference comprises a P atthe position corresponding to position 388 of SEQ ID NO: 1.

In some embodiments, the thermophilic DNA polymerase comprises an aminoacid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to asequence selected from SEQ ID NOs: 11 to 14, 19 to 22, 27 to 30, and 76to 79. In some embodiments, the thermophilic DNA polymerase comprises anamino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identityto SEQ ID NO: 11. In some embodiments, the thermophilic DNA polymerasecomprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or100% identity to SEQ ID NO: 12. In some embodiments, the thermophilicDNA polymerase comprises an amino acid sequence having at least 90%,95%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In some embodiments,the thermophilic DNA polymerase comprises an amino acid sequence havingat least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 14. In someembodiments, the thermophilic DNA polymerase comprises an amino acidsequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ IDNO: 19. In some embodiments, the thermophilic DNA polymerase comprisesan amino acid sequence having at least 90%, 95%, 98%, 99%, or 100%identity to SEQ ID NO: 20. In some embodiments, the thermophilic DNApolymerase comprises an amino acid sequence having at least 90%, 95%,98%, 99%, or 100% identity to SEQ ID NO: 21. In some embodiments, thethermophilic DNA polymerase comprises an amino acid sequence having atleast 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 22. In someembodiments, the thermophilic DNA polymerase comprises an amino acidsequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ IDNO: 27. In some embodiments, the thermophilic DNA polymerase comprisesan amino acid sequence having at least 90%, 95%, 98%, 99%, or 100%identity to SEQ ID NO: 28. In some embodiments, the thermophilic DNApolymerase comprises an amino acid sequence having at least 90%, 95%,98%, 99%, or 100% identity to SEQ ID NO: 29. In some embodiments, thethermophilic DNA polymerase comprises an amino acid sequence having atleast 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 30. In someembodiments, the thermophilic DNA polymerase comprises an amino acidsequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ IDNO: 76. In some embodiments, the thermophilic DNA polymerase comprisesan amino acid sequence having at least 90%, 95%, 98%, 99%, or 100%identity to SEQ ID NO: 77. In some embodiments, the thermophilic DNApolymerase comprises an amino acid sequence having at least 90%, 95%,98%, 99%, or 100% identity to SEQ ID NO: 78. In some embodiments, thethermophilic DNA polymerase comprises an amino acid sequence having atleast 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 79.

In some embodiments, the polymerase comprises an affinity purificationtag. In some embodiments, the affinity purification tag comprises asequence of histidines, such as 6, 7, 8, 9, or 10 consecutivehistidines. The affinity purification tag can be located, e.g., at the Nor C terminus of a polypeptide of the polymerase.

Hot Start Enzymes and Compositions

In some embodiments, a polymerase according to this disclosure isprovided as a hot-start enzyme or a hot start composition. Fordiscussion of hot-start enzymes and/or compositions, see, e.g., U.S.Pat. Nos. 5,338,671; 7,074,556; US Publication 2015/0044683; USPublication 2014/0099644. As used herein, the term “hot start” generallyrefers to a means of limiting the availability of an essential reactioncomponent (e.g., a polymerase) when the reaction mixture is maintainedat a first temperature (typically a lower temperature) until a secondtemperature (typically a higher temperature) is reached which allows theessential component to participate in the reaction. Hot start reactionstypically involve incubation at a first (e.g., lower) temperature andsubsequent elevation to a second (e.g., higher) temperature which allowsthe desired reaction to take place. Activation of the hot start reactionis preferably achieved by an incubation at a temperature which is equalto or higher than the primer hybridization (annealing) temperature usedin the amplification reaction to ensure primer binding specificity. Thelength of incubation required to recover enzyme activity depends on thetemperature and pH of the reaction mixture and on the stability of theenzyme. A wide range of incubation conditions are usable; optimalconditions may be determined empirically for each reaction.

As used herein, the term “dual hot start reaction mixture” refers to thecombination of reagents or reagent solutions which are used to blocknucleic acid polymerase extension at low temperatures (e.g., ambienttemperature) until the hot start conditions of the initial denaturationtemperature in an amplification reaction (e.g., PCR) are reached. At theelevated amplification temperature, the nucleic acid polymerase is nolonger inhibited and allows for primer extension. As used herein, thedual hot start reaction mixture is meant to include a reaction mixturethat comprises at least two different mechanisms for hot start.Accordingly, “dual hot start reaction mixtures” may include more thantwo hot start mechanisms (e.g., “triple hot start reaction mixture”,“quadruple hot start reaction mixture”, “quintuple hot start reactionmixture”, and so on).

Nonlimiting exemplary hot start mechanisms include, but are not limitedto, antibodies or combinations of antibodies that block nucleic acidpolymerase activity at lower temperatures and which dissociate from thepolymerase at elevated temperatures (see, e.g., Eastlund et al.,LifeSci. Quarterly 2:2 (2001), Mizuguchi et al., J. Biochem. (Tokyo)126:762 (1999)); affibodies (small synthetic protein molecules that havehigh binding affinity for a target protein) or combinantions ofaffibodies, sometimes referred to as antibody mimetics; oligonucleotidesthat block nucleic acid polymerase activity at lower temperatures andwhich dissociate from the polymerase at elevated temperatures (see,e.g., Dang et al., J. Mol. Biol. 264:268 (1996)); reversible chemicalmodification of the nucleic acid polymerase such that the nucleic acidpolymerase activity is blocked at lower temperatures and themodifications reverse or dissociate at elevated temperatures (see, e.g.,U.S. Pat. No. 5,773,258 and Moretti et al., Biotechniques 25:716(1998)); amino acid mutations of the nucleic acid polymerase thatprovide reduced activity at lower temperatures (see, e.g., Kermekchievet al., Nucl. Acids Res. 31:6139 (2003)); nucleic acid polymerase fusionproteins including hyperstable DNA binding domains and topoisomerases(see, e.g., Pavlov et al., Proc. Natl. Acad. Sci. USA 99:13510 (2002));ligands that inhibit the nucleic acid polymerase in atemperature-dependent manner (for example, HotMaster™ Taq DNA polymerasefrom Eppendorf (Hauppauge, N.Y.) and 5 PRIME (Gaithersburg, Md.));single-stranded binding proteins that sequester primers at lowtemperatures (see, e.g., U.S. Patent Application Publication No.2008/0138878); thermostable pyrophosphatase which hydrolyzes inorganicpyrophosphate at elevated temperatures (see, e.g., U.S. PatentApplication Publication No. 2006/0057617); thermolabile blockers, suchas a polymerase blocking protein (see, e.g., U.S. Patent ApplicationPublication No. 2007/0009922); primer competitor sequences (see, e.g.,Puskas et al., Genome Res. 5:309 (1995) and Vestheim et al., Front.Zool. 5:12 (2008)); modified primer constructs (see, e.g., Ailenberg etal., Biotechniques 29:22 (2000) and Kaboev et al., Nucl. Acids Res.28:E94 (2000)); modified primers that improve hybridization selectivity(see, e.g., U.S. Pat. Nos. 6,794,142 and 6,001,611); primers with 3′modifications that are removable by 3′-5′ exonuclease activity (see,e.g., U.S. Patent Application Publication No. 2003/0119150 and U.S. Pat.No. 6,482,590); primers with modified nucleobases that are removable byUV irradiation (see, e.g., Young et al., Chem. Commun. (Camb) 28:462(2008)); primer modifications that are removable by thermal deprotection(see, e.g., U.S. Patent Application Publication No. 2003/0162199 andLebedev et al., Nucl. Acids Res. 36:e131 (2008)); or modification of thedNTPs with thermolabile modification groups (see, e.g., U.S. PatentApplication Publication No. 2003/0162199 and Koukhareva et al., Nucl.Acids Symp. Ser. (Oxford), 259 (2008)). Agents that are used as hotstart mechanisms, such as antibodies, oligonucleotides, affibodies,chemical modifications, etc., may be referred to as “hot startinhibitors.”

In some embodiments, the hot start composition comprises an antibodyspecific for the polymerase. In some embodiments, the hot startcomposition comprises an antibody specific for the polymerase, which isbound to the polymerase. In some embodiments, the hot start compositioncomprises an inhibitor specific for the polymerase, which is bound tothe polymerase. In some embodiments, the inhibitor comprises anAffibody®. Affibodies are described, e.g., in US Publication2012/0082981; see also Nord et al., 2000, J. Biotechnol. 80: 45-54; U.S.Pat. No. 6,602,977; Nygren, 2008, FEBS J. 275: 2668-2676; Nord et al.,1997, 15: 772-777; U.S. Pat. No. 5,831,012. In some embodiments, theinhibitor comprises an oligonucleotide. In some embodiments, theinhibitor comprises a chemical modification.

As used herein, dual hot start reaction mixtures comprising “at leasttwo different mechanisms” encompass those reaction mixtures that maycomprise at least two different hot start mechanisms that functionsimilarly or use similar components. For example, dual hot startreaction mixtures can comprise reagents or reagent solutions designedfor two different antibody-based hot start mechanisms, or two differentoligonucleotide-based hot start mechanisms, or one antibody-based andone oligonucleotide-based hot start mechanism, or one antibody-based andone chemical modification-based hot start mechanism, or any suchcombination available.

Hot Start Antibodies and Methods of Making Same

In some embodiments, a hot start composition or dual hot startcomposition comprises an antibody inhibitor of a thermostable polymerasedescribed herein. In some embodiments, the antibody is a monoclonalantibody.

Methods for producing and screening for antibodies that are suitable foruse in hot start compositions with the polymerases described herein areknown in the art. In some embodiments, a hot start antibody inhibits thenucleic acid synthesis activity of the thermostable polymerase describedherein. In some embodiments, a hot start antibody inhibits exonucleaseactivity of the thermostable polymerase. In some embodiments, a hotstart antibody inhibits both the nucleic acid synthesis activity and theexonuclease activity of the thermostable polymerase.

In some embodiments, hot-start antibodies increase the specificity ofnucleic acid synthesis reactions, because they inactivate the polymeraseat room temperature, thus avoiding extension of nonspecificallyhybridized primers. The functional activity of the polymerase isrestored by disassociating the antibody from the polymerase, forexample, by incubating the composition at a higher temperature. In someembodiment, the “higher temperature” is from about 65° C. to about 99°C., from about 70° C. to about 99° C., 75° C. to about 99° C., or fromabout 80° C. to about 99° C., or from about 85° C. to about 99° C., orfrom about 90° C. to about 99° C., for a time period of at least 15seconds, or at least 30 seconds, or at least 1 minute, or at least 90seconds, or at least 2 minutes; to about 3 minutes, or about 4 minutes,or about 5 minutes, or about 7 minutes, or about 10 minutes, or more. Insome embodiments, the higher temperature is at least 60° C., at least65° C., at least 70° C., at least 75° C., at least 80° C., or at least85° C. In some embodiments, the temperature and duration of incubationto disassociate the antibody and activate the polymerase may bedetermined for the particular polymerase and antibody to be employed.

Methods for screening for antibodies of use in the present inventioninclude methods known in the art, such as affinity-based ELISA assays,as well as functional assays for polymerase and/or exonucleaseinhibition. For such functional assays, the amount of DNA produced ordigested per unit of time can be correlated to the activity of thepolymerase or exonuclease used, thus providing an estimate of the amountof inhibition a particular antibody can exert on either or both thepolymerase and exonuclease activity of the polymerase.

Antibodies may be produced using any method known in the art. As anon-limiting example, an antibody to a particular antigen (such as apolymerase described herein) may be produced by immunizing an animal(such as a mouse, rat, rabbit, goat, sheep, horse, etc.) with theantigen and isolating antibodies from the serum of the animal and/orimmortalizing primary B cells from the animal to produce hybridomas thatexpress the antibodies. Phage display technology may also be used toproduce antibodies that bind to the polymerases described herein. Phagedisplay libraries are commercially available and methods of selectingantibodies from such libraries are known in the art. See, e.g., Vaughanet al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998,Proc. Natl. Acad. Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991,J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581.Exemplary assays to determine polymerase processivity, yield,sensitivity, and specificity

Polymerase processivity may be measured using various methods known inthe art. In some embodiments, processivity refers to the number ofnucleotides incorporated during a single binding event of polymerase toa primed template. As a nonlimiting example, a detectably labeled primermay be annealed to circular or linearized DNA to form a primed nucleicacid template. In measuring processivity, the primed nucleic acidtemplate may be present in significant molar excess to the polymerase toreduce the likelihood that any one primed template will be extended morethan once by a polymerase. A “significant molar excess” may be, forexample, a ratio of 500:1, or 1000:1, or 2000:1, or 4000:1, or 5000:1(primed DNA:DNA polymerase), etc., in the presence of suitable buffersand dNTPs. Nucleic acid synthesis may be initiated by adding, forexample, MgCl₂. Nucleic acid synthesis reactions are quenched at varioustimes after initiation, and analyzed by any appropriate method todetermine the length of the product. At a polymerase concentration wherethe median product length does not change with time or polymeraseconcentration, the length corresponds to the processivity of the enzyme.In some embodiments, the processivity of a polymerase described, such asa polymerase comprising a neutral amino acid at position correspondingto position 762 of SEQ ID NO: 1 may be compared to the processivity ofthe same polymerase without the neutral amino acid mutation.

In some embodiments, yield can be demonstrated by measuring the abilityof a polymerase to produce product. Increased yield can be demonstratedby determining the amount of product obtained in a reaction using apolymerase described herein (such as a polymerase comprising a neutralamino acid at position corresponding to position 762 of SEQ ID NO: 1),as compared to the amount of product obtained in a reaction carried outunder the same reaction conditions, but with the same polymerase withoutthe neutral amino acid mutation.

In some embodiments, long PCR may be used to determine enhancedprocessivity and yield. For example, an enzyme with enhancedprocessivity typically allows the amplification of a longer amplicons(>5 kb) in shorter extension times compared to an enzyme with relativelylower processivity.

Other methods of assessing efficiency of the polymerases of theinvention can be determined by those of ordinary skill in the art usingstandard assays of the enzymatic activity of a given modificationenzyme.

The sensitivity of a polymerase described herein may be determined bymeasuring the yield of nucleic acid synthesis product in a series ofreactions with differing copy numbers of nucleic acid template. In someembodiments, the template copy number at which a polymerase of theinvention (such as a polymerase comprising a neutral amino acid atposition corresponding to position 762 of SEQ ID NO: 1) producesdetectable product is compared to the template copy number at which thesame polymerase without the neutral amino acid mutation producesdetectable product. The lower the template copy number at which thepolymerase produces detectable product, the more sensitive thepolymerase.

In some embodiments, specificity of a polymerase may be measured bydetermining the ability of the polymerase to discriminate betweenmatched primer/template duplexes and mismatched primer/templateduplexes. In some embodiments, specificity is a measure of thedifference in the relative yield of two reactions, one of which employsa matched primer, and one of which employs a mismatched primer. In someembodiments, an enzyme with increased discrimination will have a higherrelative yield with the matched primer than with the mismatched primer.In some embodiments, a ratio of the yield with the matched primer versusthe mismatched primer is determined. In some embodiments, the ratio canbe compared to the yield obtained under the same reaction conditionsusing the parental polymerase.

DNA synthesis methods; kits, compositions, systems, and apparatuses.

Provided herein are methods of synthesizing or amplifying DNA andrelated kits, compositions, systems, and apparatuses involving at leastone polymerase according to this disclosure. In some embodiments,reagents for nucleic acid synthesis are provided. In some embodiments,reagents for nucleic acid synthesis include any one or any combinationof target polynucleotides, particles attached with capture primers,solution-phase primers, fusion primers, other additional primers,enzymes (e.g., polymerases), accessory proteins (e.g., recombinase,recombinase loading protein, single-stranded binding protein, helicaseor topoisomerase), nucleotides, divalent cations, binding partners,co-factors and/or buffer. In some embodiments, reagents for nucleic acidsynthesis include a dUTPase as an accessory protein.

In some embodiments, the disclosure relates generally to compositions,as well as related systems, methods, kits and apparatuses, comprisingone or more nucleotides. In some embodiments, the compositions (andrelated methods, systems, kits and apparatuses) includes one type, or amixture of different types of nucleotides. A nucleotide comprises anycompound that can bind selectively to, or can be polymerized by, apolymerase. Typically, but not necessarily, selective binding of thenucleotide to the polymerase is followed by polymerization of thenucleotide into a nucleic acid strand by the polymerase. Suchnucleotides include not only naturally occurring nucleotides but alsoany analogs, regardless of their structure, that can bind selectivelyto, or can be polymerized by, a polymerase. While naturally occurringnucleotides typically comprise base, sugar and phosphate moieties, thenucleotides of the present disclosure can include compounds lacking anyone, some or all of such moieties. In some embodiments, the nucleotidecan optionally include a chain of phosphorus atoms comprising three,four, five, six, seven, eight, nine, ten or more phosphorus atoms. Insome embodiments, the phosphorus chain can be attached to any carbon ofa sugar ring, such as the 5′ carbon. The phosphorus chain can be linkedto the sugar with an intervening 0 or S. In some embodiments, one ormore phosphorus atoms in the chain can be part of a phosphate grouphaving P and O. In some embodiments, the phosphorus atoms in the chaincan be linked together with intervening O, NH, S, methylene, substitutedmethylene, ethylene, substituted ethylene, CNH2, C(O), C(CH2), CH2CH2,or C(OH)CH2R (where R can be a 4-pyridine or 1-imidazole). In someembodiments, the phosphorus atoms in the chain can have side groupshaving 0, BH3, or S. In the phosphorus chain, a phosphorus atom with aside group other than 0 can be a substituted phosphate group. In thephosphorus chain, phosphorus atoms with an intervening atom other than 0can be a substituted phosphate group. Some examples of nucleotideanalogs are described in Xu, U.S. Pat. No. 7,405,281.

Some examples of nucleotides that can be used in the disclosedcompositions (and related methods, systems, kits and apparatuses)include, but are not limited to, ribonucleotides, deoxyribonucleotides,modified ribonucleotides, modified deoxyribonucleotides, ribonucleotidepolyphosphates, deoxyribonucleotide polyphosphates, modifiedribonucleotide polyphosphates, modified deoxyribonucleotidepolyphosphates, peptide nucleotides, modified peptide nucleotides,metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, analogs, derivatives, or variantsof the foregoing compounds, and the like. In some embodiments, thenucleotide can comprise non-oxygen moieties such as, for example, thio-or borano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof. In some embodiments, a nucleotide can include apurine or pyrimidine base, including adenine, guanine, cytosine,thymine, uracil or inosine. In some embodiments, a nucleotide includesdATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the nucleotide is unlabeled. In some embodiments,the nucleotide comprises a label and referred to herein as a “labelednucleotide”. In some embodiments, the label can be in the form of afluorescent dye attached to any portion of a nucleotide including abase, sugar or any intervening phosphate group or a terminal phosphategroup, i.e., the phosphate group most distal from the sugar.

In some embodiments, the disclosure relates generally to compositions,as well as related systems, methods, kits and apparatuses, comprisingany one or any combination of capture primers, reverse solution-phaseprimers, fusion primers, target polynucleotides and/or nucleotides thatare non-labeled or attached to at least one label. In some embodiments,the label comprises a detectable moiety. In some embodiments, the labelcan generate, or cause to generate, a detectable signal. In someembodiments, the detectable signal can be generated from a chemical orphysical change (e.g., heat, light, electrical, pH, salt concentration,enzymatic activity, or proximity events). For example, a proximity eventcan include two reporter moieties approaching each other, or associatingwith each other, or binding each other. In some embodiments, thedetectable signal can be detected optically, electrically, chemically,enzymatically, thermally, or via mass spectroscopy or Ramanspectroscopy. In some embodiments, the label can include compounds thatare luminescent, photoluminescent, electroluminescent, bioluminescent,chemiluminescent, fluorescent, phosphorescent or electrochemical. Insome embodiments, the label can include compounds that are fluorophores,chromophores, radioisotopes, haptens, affinity tags, atoms or enzymes.In some embodiments, the label comprises a moiety not typically presentin naturally occurring nucleotides. For example, the label can includefluorescent, luminescent or radioactive moieties.

In some embodiments, the nucleic acid synthesis reaction includes acycled amplification reaction, such as a polymerase chain reaction (PCR)(U.S. Pat. Nos. 4,683,195 and 4,683,202 both granted to Mullis).Multiple examples of PCR according to this disclosure are providedbelow. In some embodiments, the nucleic acid synthesis reaction includesan isothermal reaction, such as an isothermal self-sustained sequencereaction (Kwoh 1989 Proceedings National Academy of Science USA86:1173-1177; WO 1988/10315; and U.S. Pat. Nos. 5,409,818, 5,399,491,and 5,194,370), or a recombinase polymerase amplification (RPA) (U.S.Pat. No. 5,223,414 to Zarling, U.S. Pat. Nos. 5,273,881 and 5,670,316both to Sena, and U.S. Pat. Nos. 7,270,981, 7,399,590, 7,435,561,7,666,598, 7,763,427, 8,017,339, 8,030,000, 8,062,850, and 8,071,308).

PCR is a nucleic acid synthesis reaction in which the reaction mixtureis subjected to reaction cycles, each reaction cycle comprising adenaturation period and at least one annealing and/or extension period,resulting if successful in synthesis of copies of a nucleic acidtemplate in at least the initial cycles, and copies of the copies in atleast the later cycles, generally resulting in exponential amplificationof the template. In PCR, in some instances, a pair of primers areprovided that bind at each end of a target region, on opposite strandssuch that they each prime synthesis toward the other primer. Thereaction is thermocycled so as to drive denaturation of the substrate ina high temperature step, annealing of the primers at a lower temperaturestep, and extension at a temperature which may be but is not necessarilyhigher than that of the annealing step. Exponential amplification occursbecause the products of one cycle can serve as template in the nextcycle.

An embodiment of isothermal self-sustained sequence reaction, alsosometimes referred to as transcription-mediated amplification or TMA,involves synthesizing single-stranded RNA, single-stranded DNA anddouble-stranded DNA. The single-stranded RNA is a first template for afirst primer, the single-stranded DNA is a second template for a secondprimer, and the double stranded DNA is a third template for synthesis ofa plurality of copies of the first template. A sequence of the firstprimer or the second primer is complementary to a sequence of a targetnucleic acid and a sequence of the first primer or the second primer ishomologous to a sequence of the target nucleic acid. In an embodiment ofan isothermal self-sustained sequence reaction, a first cDNA strand issynthesized by extension of the first primer along the target by anenzyme with RNA-dependent DNA polymerase activity, such as a reversetranscriptase. The first primer can comprise a polymerase bindingsequence (PBS) such as a PBS for a DNA-dependent RNA polymerase, such asT7, T3, or SP6 RNA polymerase. The first primer comprising a PBS issometimes referred to as a promoter-primer. The first cDNA strand isrendered single-stranded, such as by denaturation or by degradation ofthe RNA, such as by an RNase H. The second primer then anneals to thefirst cDNA strand and is extended to form a second cDNA strand by a DNApolymerase activity. Forming the second cDNA strand renders the cDNAdouble-stranded, including the PBS. RNA can then be synthesized from thecDNA, which comprises the PBS, by a DNA-dependent RNA polymerase, suchas T7, T3, or SP6 RNA polymerase, thereby providing a template forfurther events (extension of the first primer, rendering the productsingle-stranded, extension of the second primer, and RNA synthesis).Exponential amplification occurs because the RNA product cansubsequently serve as a template and also because RNA products can begenerated repeatedly from a cDNA comprising the PBS.

An embodiment of RPA can be performed isothermally and employs arecombinase to promote strand invasion of a double-stranded template byforward and reverse primers. The 3′ ends of the primers are extended,displacing template strands at least in part. Subsequent strandinvasion/annealing events, including to previously produced extensionproducts, occur and are followed by extension, resulting inamplification. In some embodiments, recombinase activity is supported bythe presence of one or more recombinase accessory proteins, such as arecombinase loading protein and/or single-stranded binding protein.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a nucleicacid synthesis reaction (synthesis condition) that can be conductedunder thermocycling or isothermal conditions, or a combination of bothtypes of conditions. For example, the synthesis condition can includealternating between thermocycling and isothermal synthesis conditions,in any order.

In some embodiments thermocycling synthesis conditions comprise anucleic acid synthesis reaction mixture that is subjected to an elevatedtemperature for a period of time that is sufficient to denature at leastabout 30-95% of the double-stranded target nucleic acids, and thensubjected to a lower temperature for a period of time that is sufficientto permit hybridization between the single-stranded target nucleic acidsand any of the primers (e.g., capture primer, reverse solution-phaseprimer, or fusion primer). In some embodiments, the increase anddecrease temperature cycle is repeated at least once.

In some embodiments isothermal synthesis conditions comprise a nucleicacid synthesis reaction mixture that is subjected to a temperaturevariation which is constrained within a limited range during at leastsome portion of the synthesis, including for example a temperaturevariation is within about 20° C., or about 10° C., or about 5° C., orabout 1-5° C., or about 0.1-1° C., or less than about 0.1° C.

In some embodiments, an isothermal nucleic acid synthesis reaction canbe conducted for about 2, 5, 10, 15, 20, 30, 40, 50, 60 or 120 minutes,or longer. In some embodiments, an isothermal nucleic acid synthesisreaction can be conducted for at least about 2 minutes. In someembodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 120 minutes or less. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 2to about 120 minutes. In some embodiments, an isothermal nucleic acidsynthesis reaction can be conducted for about 2 to about 60 minutes. Insome embodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 60 to about 120 minutes. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 2to about 5 minutes. In some embodiments, an isothermal nucleic acidsynthesis reaction can be conducted for about 5 to about 10 minutes. Insome embodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 10 to about 15 minutes. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 10to about 15 minutes. In some embodiments, an isothermal nucleic acidsynthesis reaction can be conducted for about 10 to about 15 minutes. Insome embodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 15 to about 20 minutes. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 20to about 30 minutes. In some embodiments, an isothermal nucleic acidsynthesis reaction can be conducted for about 30 to about 40 minutes. Insome embodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 40 to about 50 minutes. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 50to about 60 minutes.

In some embodiments, an isothermal nucleic acid synthesis reaction canbe conducted at about 15-30° C., or about 30-45° C., or about 45-60° C.,or about 60-75° C., or about 75-90° C., or about 90-93° C., or about93-99° C.

In some embodiments, the disclosure relates generally to methods, andrelated compositions, systems, kits and apparatuses, that furtherinclude an enrichment step. In some embodiments, an enrichment stepcomprises a pre-amplification reaction. See, e.g., U.S. Pat. No.8,815,546 B2. As a nonlimiting example, a pre-amplification reaction maycomprise random primers to amplify a portion, even a substantialportion, of the nucleic acid template in a sample. In this manner, theoverall amount of nucleic acid template may be increased prior to asequence-specific nucleic acid synthesis reaction.

In some embodiments, an amplified population of nucleic acids caninclude an affinity moiety. For example, in conducting any of thenucleic acid synthesis methods according to the present teachings, asolution-phase/reverse primer that is attached to an affinity moiety(e.g., biotin) can be used to conduct a synthesis reaction to produce anamplified population of nucleic acids that are attached to the affinitymoiety. In some embodiments, the enrichment step comprises forming aenrichment complex by binding the affinity moiety (which is attached tothe amplified population of nucleic acids) with a purification particle(e.g., paramagnetic bead) that is attached to a receptor moiety (e.g.,streptavidin). An example of purification particles include MyOne™ Beadsfrom Dynabeads, which are paramagnetic beads attached to streptavidin.In some embodiments, a magnet can be used to separate/remove theenrichment complex from amplified population of nucleic acids that lackthe affinity moiety. In some embodiments, the enrichment step can berepeated at least once. In some embodiment, the enrichment step isfollowed by one or more washing step.

In some embodiments, the disclosure relates generally to methods, andrelated compositions, systems, kits and apparatuses that further includeat least one washing step. The washing step can be conducted at any timeduring the workflow for nucleic acid synthesis. In some embodiments, awashing step can remove excess or unreacted components of the nucleicacid synthesis or enrichment reactions.

In some embodiments, any of the nucleic acid synthesis methods, orenrichment steps, according to the present teachings, can be conductedmanually or by automation. In some embodiments, any one or anycombination of the steps can be conducted manually or by automation,including: conducting a nucleic acid synthesis reaction, enriching,and/or washing. For example, any reagents for a nucleic acid synthesis,enrichment or washing, can be deposited into, or removed from, areaction vessel via manual or automated modes.

In various embodiments, the disclosure relates to compositionscomprising at least one polymerase described herein. In someembodiments, the composition is a hot start composition. In some suchembodiments, the composition is a dual hot start composition. In someembodiments, the dual hot start composition comprises at least twodifferent hot start mechanisms that are used to inhibit or substantiallyinhibit the polymerase activity at a first temperature. Such hot startmechanisms include, but are not limited to, antibodies or combinationsof antibodies that block DNA polymerase activity at lower temperatures,antibody mimetics or combinations of antibody mimetics that block DNApolymerase activity at lower temperatures (such as, for example,Affibodies®), oligonucleotides that block DNA polymerase activity atlower temperatures (such as, for example, aptamers), reversible chemicalmodifications of the DNA polymerase that dissociate at elevatedtemperatures, amino acid modifications of the DNA polymerase thatprovide reduced activity at lower temperatures, fusion proteins thatinclude hyperstable DNA binding domains and topoisomerase, othertemperature dependent ligands that inhibit the DNA polymerase, singlestranded binding proteins that sequester primers at lower temperatures,modified primers or modified dNTPs. Hot start compositions, in someembodiments, comprise at least one polymerase described herein with orwithout a hot start chemical modification, at least one hot startantibody, at least one hot start aptamer, and/or at least one hot startAffibody®. In some embodiments, a hot start composition comprises atleast one polymerase described herein with or without a hot startchemical modification, at least one hot start antibody and at least onehot start aptamer or at least one hot start Affibody®. In someembodiments, a hot start composition comprises at least one polymerasedescribed herein with or without a hot start chemical modification, atleast one hot start Affibody® and at least one hot start antibody or atleast one hot start aptamer. In some embodiments, a hot startcomposition comprises a polymerase described herein with or without ahot start chemical modification, a hot start antibody, and a hot startaptamer or a hot start Affibody®. In some embodiments, a hot startcomposition comprises a polymerase described herein with or without ahot start chemical modification, a hot start Affibody®, and a hot startantibody or a hot start aptamer. In some embodiments, a hot startcomposition comprises a polymerase described herein with or without ahot start chemical modification, a hot start antibody, and a hot startAffibody®. In some embodiments, a hot start composition comprises apolymerase described herein with or without a hot start chemicalmodification, a hot start antibody, and a hot start aptamer.

In some embodiments, a composition comprises one or more detergents, oneor more protein stabilizers, and/or at least one UTPase. In someembodiments, a composition comprises one or more detergents, one or moreprotein stabilizers, and at least one UTPase. In some embodiments, acomposition comprises at least one monovalent cationic salt, at leastone divalent cationic salt, and/or at least one dNTP. In someembodiments, a composition further comprises at least one dye. In someembodiments, a composition comprises additional stabilizers thatincrease the density of the composition.

Nonlimiting exemplary detergents that may be used in the compositionsprovided herein include nonionic, ionic (anionic, cationic) andzwitterionic detergents. Exemplary such detergents include, but are notlimited to, Hecameg(6-O—(N-Heptylcarbamoyl)-methyl-α-D-glucopyranoside), Trition X-200,Brij-58, CHAPS, n-Dodecyl-b-D-maltoside, NP-40, sodium dodecyl sulphate(SDS), TRITON® X-15, TRITON® X-35, TRITON® X-45, TRITON® X-100, TRITON®X-102, TRITON® X-114, TRITON® X-165, TRITON® X-305, TRITON® X-405,TRITON® X-705, Tween® 20 and/or ZWITTERGENT®. Other detergents may alsobe suitable, as may be determined by one of skill in the art. See, e.g.,U.S. Pat. No. 7,972,828B2, U.S. Pat. No. 8,980,333B2 U.S. PublicationNo. 2008/0145910; U.S. Publication No. 2008/0064071; U.S. Pat. Nos.6,242,235; 5,871,975; and 6,127,155 for exemplary detergents.

Nonlimiting exemplary protein stabilizers that may be used in thecompositions provided herein include BSA, inactive polymerases (such asinactivated Taq polymerase; see, e.g., US Publication No. 2011/0059490),and apotransferrin. Further nonlimiting exemplary stabilizers that maybe used in the compositions provided herein include glycerol, trehalose,lactose, maltose, galactose, glucose, sucrose, dimethyl sulfoxide(DMSO), polyethylene glycol, and sorbitol.

Nonlimiting exemplary UTPases that may be used in the compositionsprovided herein include UTPases from thermophilic bacteria. See, e.g.,PNAS, 2002, 99: 596-601.

Nonlimiting exemplary dyes that may be used in the compositions providedherein include xylene cyanol FF, tartrazine, phenol red, quinolineyellow, zylene cyanol, Brilliant Blue, Patent Blue, indigocarmine, acidred 1, m-cresol purple, cresol red, neutral red, bromocresol green, acidviolet 5, bromo phenol blue, and orange G (see, e.g., U.S. Pat. No.8,663,925 B2). Additional nonlimiting exemplary dyes are described,e.g., in U.S. Pat. No. 6,942,964. One skilled in the art will appreciatethat any dye that does not inhibit nucleic acid synthesis by thepolymerases described herein may be used.

In some embodiments, a storage composition is provided comprising apolymerase provided herein, at least one hot start antibody, at leastone protein stabilizer, and at least one UTPase, in a buffer suitablefor storage. In some embodiments, a storage composition is providedcomprising a polymerase provided herein, at least one hot startantibody, at least one Affibody®, at least one protein stabilizer, andat least one UTPase, in a buffer suitable for storage. In someembodiments, a storage composition is provided comprising a polymeraseprovided herein, two hot start antibodies, a protein stabilizer, and aUTPase, in a buffer suitable for storage. In some embodiments, thestorage buffer comprises a buffering agent (such as Tris HCl), a salt(such as KCl or NaCl), a stabilizer (such as glycerol), a reducing agent(such as DTT), a divalent cation chelating agent (such as EDTA), and adetergent (such as hecameg and/or Triton X-200 and/or NP-40 and/orTween-20, etc.). In some embodiments, the storage composition comprises0.5 to 5 units (U), or 0.5 to 3 U, or 1 to 3 U, or 2 U of polymerase perμl. In some embodiments, the storage composition comprises 0.05 to 1mg/ml, or 0.05 to 0.5 mg/ml, or 0.1 to 0.5 mg/ml, or 0.1 to 0.3 mg/ml ofeach hot start antibody. In some embodiments, the storage compositioncomprises 0.1 to 10 mg/ml, or 0.1 to 5 mg/ml, or 0.5 to 5 mg/ml, or 0.5to 2 mg/ml of each hot start Affibody®. In some embodiments, the storagecomposition comprises 0.5 to 5 mg/ml, or 1 to 5 mg/ml, or 1 to 3 mg/mlof each protein stabilizer.

In some embodiments, a reaction composition is provided, comprising atleast one polymerase described herein, at least one buffering agent(such as Tris HCl), at least one monovalent cationic salt (such as KClor NaCl), at least one divalent cationic salt (such as MgCl₂ or MnCl₂),at least one detergent (such as hecameg and/or Triton X-200 and/or NP-40and/or Tween-20, etc.), and at least one dNTP. In some embodiments, thecomposition comprises dATP, dCTP, dGTP, and dTTP. In some embodiments,the reaction composition further comprises at least one dye. In someembodiments, for example when the composition is to be loaded on a gel,the reaction composition comprises additional stabilizers that increasethe density of the composition, such as polyethylene glycol (e.g., PEG4000) and/or sucrose. PEG 4000 may be included, in some embodiments, ata concentration of 0.5-2%, or about 1%; and sucrose may be included, insome embodiments, at a concentration of 1-5%, or 1-3%, or about 2% (or2-10%, or 2-6%, or about 4% for a 2× reaction composition). In someembodiments, the buffering agent (such as Tris HCl) is present at aconcentration of 5-50 mM, or 5-30 mM, or 5-20 mM (or 10-100 mM, or 10-60mM, or 10-40 mM for a 2× reaction composition). In some embodiments, themonovalent cation (such as K+ or Na+) is present at a concentration of50-300 mM, or 50-200 mM, or 75-150 mM, or about 110 mM (or 100-600 mM,or 100-400 mM, or 150-300 mM, or about 220 mM for a 2× reactioncomposition). In some embodiments, a detergent (such as hecameg and/orTriton X-200 and/or NP-40 and/or Tween-20, etc.) is present at aconcentration of 0.05-0.3%, or 0.1-0.2%, or about 0.15% (or 0.01-0.6%,or 0.2-0.4%, or about 0.3% for a 2× reaction composition). In someembodiments, the Mg²⁺ or Mn²⁺ is present at a concentration of 0.5-5 mM,or 0.5-3 mM, or about 1.5 mM (or 1-10 mM, or 1-6 mM, or about 3 mM for a2× reaction composition). In some embodiments, each dNTP is present at aconcentration of 0.05-1 mM, or 0.1-0.8 mM, or 0.1-0.6 mM, or 0.1-0.4 mM,or about 0.2 mM (or 0.1-2 mM, or 0.2-1.6 mM, or 0.2-1.2 mM, or 0.2-0.8mM, or about 0.4 mM for a 2× reaction composition).

PCR enhancing factors may also be used to improve efficiency of theamplification. As used herein, a “PCR enhancing factor” or a “PolymeraseEnhancing Factor” (PEF) refers to a complex or protein possessingpolynucleotide polymerase enhancing activity (Hogrefe et al., 1997,Strategies 10:93-96; and U.S. Pat. No. 6,183,997, both of which arehereby incorporated by references). For Pfu DNA polymerase, for example,PEF may comprise either P45 in native form (as a complex of P50 and P45)or as a recombinant protein. In the native complex of Pfu P50 and P45,only P45 exhibits PCR enhancing activity. The P50 protein is similar instructure to a bacterial flavoprotein. The P45 protein is similar instructure to dCTP deaminase and dUTPase, but it functions only as adUTPase converting dUTP to dUMP and pyrophosphate. PEF, according to thepresent disclosure, may also be selected from the group consisting of:an isolated or purified naturally occurring polymerase enhancing proteinobtained from an archaeabacteria source (e.g., Pyrococcus furiosus); awholly or partially synthetic protein having the same amino acidsequence as Pfu P45, or analogs thereof possessing polymerase enhancingactivity; polymerase-enhancing mixtures of one or more of said naturallyoccurring or wholly or partially synthetic proteins;polymerase-enhancing protein complexes of one or more of said naturallyoccurring or wholly or partially synthetic proteins; orpolymerase-enhancing partially purified cell extracts containing one ormore of said naturally occurring proteins (U.S. Pat. No. 6,183,997,supra).

In some embodiments, a reaction composition further comprisesingredients that enhance nucleic acid synthesis from high GC-contenttemplates. In some such embodiments, the reaction composition comprisesglycerol, DMSO, and/or ammonium sulphate. In some embodiments, thereaction composition comprises glycerol, DMSO, and ammonium sulphate. Insome embodiments, glycerol is present in the reaction composition at aconcentration of 5-20%, or 5-15%, or about 10%. In some embodiments,DMSO is present in the reaction composition at a concentration of 1-10%,or 3-10%, or 3-7%, or about 5%. In some embodiments, ammonium sulphateis present in the reaction composition at 10-50 mM, or 15-40 mM, or20-30 mM, or about 25 mM.

In some embodiments, a reaction composition is provided at 2×, 5×, 10×,etc. concentration, in which case, the concentrations discussed hereinare multiplied (e.g., as noted above; doubled for 2×). A 2× reactioncomposition is typically diluted by 2-fold, for example, when thetemplate nucleic acid and/or primers are added to the composition.

In some embodiments, a reaction composition comprises nucleic acidtemplate and at least one primer for nucleic acid synthesis. In someembodiments, each primer is included in the reaction composition at aconcentration of 0.1-0.8 μM, or 0.1-0.5 μM, or 0.2-0.4 μM, or about 0.3μM. One skilled in the art will appreciate that the template nucleicacid may be provided at a wide range of concentrations, which lowerlimit, in some embodiments, may be determined by the sensitivity of thepolymerase.

In some embodiments, the composition comprises at least one PCRinhibitor. In some embodiments, the PCR inhibitor comprises xylan,heparin, humic acid, or SDS. In some embodiments, methods according tothe disclosure comprise amplifying DNA in the presence of at least onePCR inhibitor. In some embodiments, the PCR inhibitor comprises xylan.In some embodiments, the PCR inhibitor comprises heparin.

In various embodiments, the composition may be an aqueous composition.In various embodiments, the composition may be a lyophilizedcomposition. In some embodiments, the composition comprises acryoprotectant and/or a preservative and/or other additives known tothose skilled in the art. Nonlimiting exemplary cryoprotectants andpreservatives include, for example, the stabilizers and reducing agentsdescribed herein. Nucleic acids; vectors; host cells; methods ofproduction and/or purification.

Provided herein are nucleic acids comprising a sequence encoding apolymerase according to this disclosure. In some embodiments, thenucleic acid is operably linked to a promoter. In some embodiments, thepromoter is a promoter for a bacteriophage RNA polymerase, such as a T7promoter. In some embodiments, the nucleic acid is codon-optimized forexpression in a host cell, such as a microorganism, e.g., a bacterium,such as E. coli.

Also provided herein are vectors comprising any of the nucleic acidscomprising a sequence encoding a polymerase according to this disclosurediscussed above. In some embodiments, the vector is a plasmid. In someembodiments, the vector is an expression vector. In some embodiments,the vector contains a selectable marker. In some embodiments, the vectoris capable of being propagated in a microorganism, e.g., a bacterium,such as E. coli.

Also provided herein are host cells comprising any of the nucleic acidscomprising a sequence encoding a polymerase according to this disclosurediscussed above. Also provided herein are host cells comprising any ofthe vectors comprising a sequence encoding a polymerase according tothis disclosure discussed above. In some embodiments, the host cell is amicroorganism, e.g., a bacterium, such as E. coli. In some embodiments,the host cell further comprises a nucleic acid encoding a heterologousRNA polymerase. In some embodiments, the heterologous RNA polymerase isa bacteriophage RNA polymerase, such as bacteriophage T7 RNA polymerase.In some embodiments, the heterologous RNA polymerase is operably linkedto a promoter, such as an inducible promoter, e.g., a lac-induciblepromoter. In some embodiments, the host cell is of a protease-deficientstrain. In some embodiments, the host cell is E. coli BL-21. In someembodiments, the host cell, such as BL-21, is modified to carry tRNAgenes encoding tRNAs with rarer anticodons (for example, argU, ileY,leuW, and proL tRNA genes).

Also provided herein are methods of producing and/or purifying apolymerase according to this disclosure. In some embodiments, such amethod comprises culturing at least one host cell comprising a nucleicacid encoding a thermophilic DNA polymerase according to thisdisclosure, wherein the at least one host cell expresses thethermophilic DNA polymerase. In some embodiments, such a methodcomprises isolating a polymerase according to this disclosure from hostcells that have expressed the polymerase. In some embodiments, theisolating comprises lysing the host cells. In some embodiments, theisolating comprises heat treatment to denature host proteins. In someembodiments, denatured host proteins are removed, e.g., bycentrifugation. In some embodiments, the polymerase is purified viachromatography. Examples of procedures for purifying DNA polymerases areprovided, e.g., in Lawyer et al. (1993, PCR Meth. & App. 2: 275)(designed originally for the isolation of Taq polymerase) and Kong etal. (1993, J. Biol. Chem. 268: 1965) (involving a heat denaturation stepof host proteins, and two column purification steps over DEAE-Sepharoseand heparin-Sepharose columns).

EXAMPLES

The following examples are provided to illustrate certain disclosedembodiments and are not to be construed as limiting the scope of thisdisclosure in any way.

Example 1. Tolerance of Inhibitors by a Thermophilic DNA PolymeraseAccording to the Disclosure

The inhibitor resistance of a thermophilic DNA polymerase with thesequence of SEQ ID NO: 20 (including a Q at position 762) (762Qpolymerase) was compared to a version with a K at position 762 (762Kpolymerase) by amplifying PCR fragments in the presence of variousamounts of polymerase inhibitors. The performance of a thermophilic DNApolymerase with the sequence of SEQ ID NO: 22 (including a Q at position762 and S at position 408) (408S 762Q polymerase) was compared to aversion with a K at position 762 (408S 762K polymerase) by amplifyingPCR fragments in the presence of various amounts of polymeraseinhibitors.

Heparin.

A 2 kb fragment was amplified from 20 ng of human genomic DNA templatein 20 μl PCR reactions in the presence of 0 to 0.3 μM of heparin usingthe thermophilic DNA polymerases (FIG. 1 ). Primers with the followingsequences were used: GAAGAGCCAAGGACAGGTAC (SEQ ID NO: 64) (forward);CCTCCAAATCAAGCCTCTAC (SEQ ID NO: 65) (reverse). The PCR program was asfollows:

98° C. for 30 s;

repeat 30 cycles of the following: 98° C. for 10 s;

-   -   60° C. for 10 s;    -   72° C. for 1 min        72° C. for 5 min.

Products were detected by agarose gel electrophoresis and staining withEthidium bromide. Detectable product was observed at up to 0.25 μMheparin for the 762Q polymerase and 0.15 μM for 408S 762Q polymerase.Products were not detected at or above 0.1 μM heparin for the 762K and408S 762K polymerases.

Xylan.

A 2 kb fragment was amplified from 40 ng of human genomic DNA templatein 20 μl PCR reactions in the presence of 0 to 400 ng/μ1 xylan using thethermophilic DNA polymerases (FIG. 2 ). The primers, PCR program, andproduct detection were as described above with respect to heparin.

Detectable product was observed at up to 400 ng/μl xylan for the 762Qpolymerase. Products were not detected at 400 ng/μl xylan for the 762Kpolymerases.

Humic acid.

A 2 kb fragment was amplified from 40 ng of human genomic DNA templatein 20 μl PCR mixture in the presence of 0 to 1.0 ng/ml of humic acidusing a thermophilic DNA polymerase with the sequence of SEQ ID NO: 20(including a Q at position 762) was compared to a version with a K atposition 762 (FIG. 3 ). Primers, the PCR program, and product detectionwere as described above with respect to heparin.

Sodium dodecyl sulfate.

A 2 kb fragment was amplified from 40 ng of human genomic DNA templatein 20 μl PCR mixture in the presence of 0 to 0.016% or 0.2% (w/v) sodiumdodecyl sulfate (SDS) using the 762Q or 762K polymerases (FIG. 4 ). Theprimers, PCR program, and product detection were as described above withrespect to heparin.

Thus, increased inhibitor tolerance was observed for the polymeraseswith a Q at position 762.

Example 2. Yield and Sensitivity of PCR with a Thermophilic DNAPolymerase According to the Disclosure

The PCR performance (sensitivity and yield) of the 762Q and 762Kpolymerases discussed in Example 1 were compared by amplifying PCRfragments from various amounts of DNA template.

Sensitivity.

A 2 kb fragment was amplified from a series of amounts of human genomicDNA template between 0 and 400 ng in a 20 μl PCR mixture using thethermophilic DNA polymerases (FIG. 5 ). The primers and the PCR programwere the same as those used in Example 1. Products were analyzed byagarose gel electrophoresis and stained as in Example 1.

The results showed that the reaction with the 762Q polymerase had highersensitivity (amplification from lower amounts of template DNA) than withthe 762K polymerase, and the reaction with the 408S 762Q polymerase hadhigher sensitivity than with the 408S 762K polymerase.

Yield A 10 kb fragment was amplified from a series of amounts of phagelambda DNA template between 0 and 200 ng in a 20 μl PCR mixture usingthe thermophilic DNA polymerases. The primers were:CAGTGCAGTGCTTGATAACAGG (SEQ ID NO: 66) (forward) andGTAGTGCGCGTTTGATTTCC (SEQ ID NO: 67) (reverse). The PCR program was: 98°C. for 30 s;

-   -   repeat 25 cycles of the following: 98° C. for 10 s;    -   60° C. for 15 s;    -   72° C. for 150 s        72° C. for 10 min.

Products were analyzed by agarose gel electrophoresis and stained as inExample 1. The results from two experiments showed that the reactionsensitivity (amplification from lower amounts of template DNA) wassimilar for all the polymerases (FIG. 6A), while the 762Q polymeraseshowed the highest yield (140% from the 762K polymerase) amplifying the10 kb fragment from 0.5 ng of the template (FIG. 6B).

Example 3. Fidelity of Thermophilic DNA Polymerases According to theDisclosure

Polymerase fidelity was measured by next generation sequencing.Fragmented E. coli DNA (˜300 bp) was amplified by Taq polymerase, 762Kpolymerase, 762Q polymerase, 408S 762K polymerase and 408S 762Qpolymerase. The number of effective PCR cycles was found by qPCR. Theamplified libraries were subjected to paired-end Illumina sequencingtogether with control E. coli PCR-free libraries. The polymerase errorrates were calculated using bioinformatics techniques. The backgroundlevel of experimental errors was estimated from PCR-free librarysequencing data. The polymerase introduced errors were identified asnucleotide changes in both pair-end reads, while nucleotide changes inonly pair-end one read have been treated as instrumental errors and wereeliminated. The polymerase fidelities (1/error rate) were normalized tothe fidelity of Taq polymerase, which fidelity value is indicated as 1×.

The fidelity of the 762K polymerase was ˜50× of the Taq polymerase, the762Q polymerase also showed similar fidelity (Table 1). The error ratesfor the 408S 762K and A408S 762Q polymerases were almostindistinguishable from the background, which indicate >100× fidelity ofthe Taq polymerase and is the threshold of fidelity measurements usingthis particular experimental setup.

TABLE 1 Polymerase Fidelity, xTaq polymerase fidelity Taq 1 x 762K 20-70x 762Q 20-70 x 408S 762K >100 x * 408S 762Q >100 x * * 100 x Taqfidelity is the threshold of fidelity measurements

Example 4. Yield and Sensitivity of PCRwith a Thermophilic DNAPolymerase According to the Disclosure Provided as Hot-StartCompositions

The performance of a thermophilic DNA polymerase with the sequence ofSEQ ID NO: 22 (including a Q at position 762 and S at position 408)(408S 762Q polymerase) was compared to a version with a K at position762 (408S 762K polymerase) by amplifying various PCR fragments, with thepolymerases being provided as dual hot-start compositions. A dUTPase wasalso supplied in the reactions.

2 kb fragment from human genomic DNA template.

The template was human genomic DNA in a series of amounts between 0 and400 ng in 20 μl reactions. The primers and the PCR program were the sameas in Example 1.

The 408S 762Q polymerase showed increased yield and higher sensitivityrelative to the 408S (FIG. 7A).

5 kb fragment from phage lambda DNA template.

PCR primers were CCTGCTCTGCCGCTTCACGC (SEQ ID NO: 68) (forward) andCGAACGTCGCGCAGAGAAACAGG (SEQ ID NO: 69) (reverse). The PCR program was:

98° C. for 30 s;

repeat 30 cycles of the following: 98° C. for 10 s;

-   -   72° C. for 1 min 40 s        72° C. for 10 min.

Lambda DNA was provided as template at amounts between 0 and 200 ng in20 μl reactions. Products were analyzed by agarose gel electrophoresisand stained as in Example 1. Sensitivity was higher for reactions withthe 408S 762Q polymerase (FIG. 7B).

20 kb fragment from phage lambda DNA template.

PCR primers were CTGATGAGTTCGTGTCCGTACAACTGGCGTAATC (SEQ ID NO: 70)(forward) and GTGCACCATGCAACATGAATAACAGTGGGTTATC (SEQ ID NO: 71)(reverse). The PCR program was:

98° C. for 30 s;

repeat 25 cycles of the following: 98° C. for 10 s;

-   -   72° C. for 10 min        72° C. for 10 min.

Lambda DNA was provided as template at amounts between 0 and 100 ng in20 μl reactions. Products were analyzed by agarose gel electrophoresisand stained as in Example 1. Band intensities from lower amounts of thetemplate were generally greater for reactions with the 408S 762Qpolymerase, indicating increased yield and sensitivity (FIG. 8 ).

20 kb fragment from Escherichia coli genomic DNA template.

PCR primers were: GGGCGTTTTCCGTAACACTG (SEQ ID NO: 72) (forward) andTGACCACATACAATCGCCGT (SEQ ID NO: 73) (reverse). The PCR program was:

98° C. for 30 s;

repeat 30 cycles of the following: 98° C. for 10 s;

-   -   61° C. for 30 s;    -   72° C. for 10 min        72° C. for 10 min.

E. coli gDNA template was provided as template at amounts between 0 and40 ng in 20 μl reactions. Products were analyzed by agarose gelelectrophoresis and stained as in Example 1.

Band intensities from lower amounts of the template were generallygreater for reactions with the 408S 762Q polymerase, indicatingincreased yield and sensitivity (FIG. 9 ).

7.5 kb fragment from human genomic DNA template.

The template was human genomic DNA in a series of amounts between 0 and400 ng in 20 μl reactions. Primers were: CTCCACAGGGTGAGGTCTAAGTGATGACA(SEQ ID NO: 74) (forward) and CAATCTCAGGGCAAGTTAAGGGAATAGTG (SEQ ID NO:75) (reverse). The PCR program was:

98° C. for 30 s;

repeat 30 cycles of the following: 98° C. for 10 s;

-   -   72° C. for 180 s        72° C. for 10 min.

Band intensities were greater for reactions with the 408S 762Qpolymerase, indicating increased yield and sensitivity (FIG. 10 ).

Example 5. Fidelity of Thermophilic DNA Polymerases Provided asHot-Start Compositions

Polymerase fidelity was measured by next generation sequencing.Fragmented E. coli DNA (˜300 bp) was amplified by Taq polymerase, 408Spolymerase, and 408S 762Q polymerase with the polymerases being providedas dual hot-start compositions, including affibodies and antibodies. AdUTPase was also supplied in the reactions. Polymerase fidelities weremeasured as in the Example 3.

The error rates for the 408S and 408S 762Q polymerases were almostindistinguishable from the background, which indicate >100× fidelity ofthe Taq polymerase and is the threshold of fidelity measurements usingthis particular experimental setup (Table 2).

TABLE 2 Polymerase Fidelity, xTaq polymerase fidelity Taq 1 x 408S >100x * 408S 762Q >100 x * * 100 x Taq fidelity is the threshold of fidelitymeasurements

Table of Sequences SEQ ID NO Description Sequence  1Pfu DNA polymerase (GenBank MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYAAcc. No. WP_011011325.1) LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPIamino acid sequence TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYLIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TRQVGLTSWL NIKKS  2Pfu GenBank WP_011011325.1 MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYAR762X amino acid sequence LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPITVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYLIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TXQVGLTSWL NIKKS;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N.  3Pfu GenBank WP_011011325.1 MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYAR762Q amino acid sequence LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPITVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYLIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TQQVGLTSWL NIKKS  4Pfu GenBank WP_011011325.1 MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYAA408S R762X amino acid LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPIsequence TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYLIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRSLY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TXQVGLTSWL NIKKS;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N.  5Pfu GenBank WP_011011325.1 MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYAA408S R762Q amino acid LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPIsequence TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYLIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRSLY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TQQVGLTSWL NIKKS  6Pfu DNA polymerase (GenBank SYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVSAcc. No. WP_011011325.1), PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEEcatalytic domain amino acid RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGYsequence AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYIDTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEYEGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIAKETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEKLAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TRQVGL 7 Pfu DNA polymerase (GenBank SYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVSAcc. No. WP_011011325.1) PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEEcatalytic domain R762X amino RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGYacid sequence AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYIDTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEYEGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIAKETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEKLAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLPAVLRILEGFG YRKEDLRYQK TXQVGL; wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N.  8Pfu DNA polymerase (GenBank SYTGGFV KEPEKGLWEN IVYLDFRSLY PSIIITHNVSAcc. No. WP_011011325.1) PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEEcatalytic domain A408S R762X RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGYamino acid sequence AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYIDTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEYEGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIAKETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEKLAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLPAVLRILEGFG YRKEDLRYQK TXQVGL; wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N.  9Pfu DNA polymerase (GenBank SYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVSAcc. No. WP_011011325.1) PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEEcatalytic domain R762Q amino RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGYacid sequence AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYIDTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEYEGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIAKETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEKLAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TQQVGL10 Pfu DNA polymerase (GenBank SYTGGFV KEPEKGLWEN IVYLDFRSLY PSIIITHNVSAcc. No. WP_011011325.1) PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEEcatalytic domain A408S R762Q RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGYamino acid sequence AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYIDTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEYEGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIAKETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEKLAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TQQVGL11 Pfu GenBank WP_011011325.1MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYA R762X with DNA bindingLLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPI domain amino acid sequenceTVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYLIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQKTXQVGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 12Pfu GenBank WP_011011325.1 MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYAR762Q with DNA binding LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPIdomain amino acid sequence TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYLIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQKTQQVGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 13Pfu GenBank WP_011011325.1 MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYAA408S R762X with DNA LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPIbinding domain amino acid TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYsequence LIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRSLY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQKTXQVGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 14Pfu GenBank WP_011011325.1 MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYAA408S R762Q with DNA LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPIbinding domain amino acid TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRYsequence LIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRSLY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQKTQQVGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 15Pyrococcus K762X catalytic SYAGGFVKEP EKGLWENIVS LDFRALYPSI IITHNVSPDTdomain amino acid sequence LNREGCRNYD VAPEVGHKFC KDFPGFIPSL LKRLLDERQKIKTKMKASQD PIEKIMLDYR QRAIKILANS YYGYYGYAKARWYCKECAES VTAWGREYIE FVWKELEEKF GFKVLYIDTDGLYATIPGGK SEEIKKKALE FVDYINAKLP GLLELEYEGFYKRGFFVTKK KYALIDEEGK IITRGLEIVR RDWSEIAKETQARVLEAILK HGNVEEAVRI VKEVTQKLSK YEIPPEKLAIYEQITRPLHE YKAIGPHVAV AKRLAAKGVK IKPGMVIGYIVLRGDGPISN RAILAEEYDP RKHKYDAEYY IENQVLPAVL RILEGFGYRK EDLRWQKTXQ TGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 16 Pyrococcus A408S K762XSYAGGFVKEP EKGLWENIVS LDFRSLYPSI IITHNVSPDT catalytic domain amino acidLNREGCRNYD VAPEVGHKFC KDFPGFIPSL LKRLLDERQK sequenceIKTKMKASQD PIEKIMLDYR QRAIKILANS YYGYYGYAKARWYCKECAES VTAWGREYIE FVWKELEEKF GFKVLYIDTDGLYATIPGGK SEEIKKKALE FVDYINAKLP GLLELEYEGFYKRGFFVTKK KYALIDEEGK IITRGLEIVR RDWSEIAKETQARVLEAILK HGNVEEAVRI VKEVTQKLSK YEIPPEKLAIYEQITRPLHE YKAIGPHVAV AKRLAAKGVK IKPGMVIGYIVLRGDGPISN RAILAEEYDP RKHKYDAEYY IENQVLPAVL RILEGFGYRK EDLRWQKTXQ TGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 17Pyrococcus K762Q catalytic SYAGGFVKEP EKGLWENIVS LDFRALYPSI IITHNVSPDTdomain amino acid sequence LNREGCRNYD VAPEVGHKFC KDFPGFIPSL LKRLLDERQKIKTKMKASQD PIEKIMLDYR QRAIKILANS YYGYYGYAKARWYCKECAES VTAWGREYIE FVWKELEEKF GFKVLYIDTDGLYATIPGGK SEEIKKKALE FVDYINAKLP GLLELEYEGFYKRGFFVTKK KYALIDEEGK IITRGLEIVR RDWSEIAKETQARVLEAILK HGNVEEAVRI VKEVTQKLSK YEIPPEKLAIYEQITRPLHE YKAIGPHVAV AKRLAAKGVK IKPGMVIGYIVLRGDGPISN RAILAEEYDP RKHKYDAEYY IENQVLPAVL RILEGFGYRK EDLRWQKTQQ TGL 18Pyrococcus 408S K762Q SYAGGFVKEP EKGLWENIVS LDFRSLYPSI IITHNVSPDTcatalytic domain amino acid LNREGCRNYD VAPEVGHKFC KDFPGFIPSL LKRLLDERQKsequence IKTKMKASQD PIEKIMLDYR QRAIKILANS YYGYYGYAKARWYCKECAES VTAWGREYIE FVWKELEEKF GFKVLYIDTDGLYATIPGGK SEEIKKKALE FVDYINAKLP GLLELEYEGFYKRGFFVTKK KYALIDEEGK IITRGLEIVR RDWSEIAKETQARVLEAILK HGNVEEAVRI VKEVTQKLSK YEIPPEKLAIYEQITRPLHE YKAIGPHVAV AKRLAAKGVK IKPGMVIGYIVLRGDGPISN RAILAEEYDP RKHKYDAEYY IENQVLPAVL RILEGFGYRK EDLRWQKTQQ TGL 76Pyrococcus DNA polymerase MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYAsequence including exonucleaseLLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI domain and catalytic domain,TVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY K762XLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRALY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TXQTGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 77Pyrococcus DNA polymerase MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYAsequence including exonucleaseLLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI domain and catalytic domain,TVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY A408S K762XLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRSLY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TXQTGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 78Pyrococcus DNA polymerase MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYAsequence including exonucleaseLLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI domain and catalytic domain,TVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY K762QLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRALY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TQQTGL 79Pyrococcus DNA polymerase MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYAsequence including exonucleaseLLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI domain and catalytic domain,TVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY A408S K762QLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRSLY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TQQTGL 19Pyrococcus DNA polymerase MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYAsequence including exonucleaseLLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI domain and DNA bindingTVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY domain, K762XLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRALY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQKTXQTGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 20Pyrococcus DNA polymerase MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYAsequence including exonucleaseLLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI domain and DNA bindingTVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY domain, K762QLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRALY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQKTQQTGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 21Pyrococcus DNA polymerase MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYAsequence including exonucleaseLLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI domain and DNA bindingTVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY domain, A408S K762XLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRSLY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQKTXQTGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 22Pyrococcus DNA polymerase MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYAsequence including exonucleaseLLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI domain and sequence non-TVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY specific DNA binding domain,LIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMI A408S K762QSYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRSLY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQKTQQTGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 23K762X variant of Deep Vent MILDADYITE DGKPIIRIFK KENGEFKVEY DRNFRPYIYADNA polymerase amino acid LLKDDSQIDE VRKITAERHG KIVRIIDAEK VRKKFLGRPIsequence EVWRLYFEHP QDVPAIRDKI REHSAVIDIF EYDIPFAKRYLIDKGLIPME GDEELKLLAF DIETLYHEGE EFAKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKVIREKDPDVIITYN GDSFDLPYLV KRAEKLGIKL PLGRDGSEPKMQRLGDMTAV EIKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YAHEIAEAWE TGKGLERVAK YSMEDAKVTYELGREFFPME AQLSRLVGQP LWDVSRSSTG NLVEWYLLRKAYERNELAPN KPDEREYERR LRESYAGGYV KEPEKGLWEGLVSLDFRSLY PSIIITHNVS PDTLNREGCR EYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQEIKRKMKA SKDPIEKKMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVRKELE EKFGFKVLYI DTDGLYATIP GAKPEEIKKKALEFVDYINA KLPGLLELEY EGFYVRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQAKVLEA ILKHGNVEEAVKIVKEVTEK LSKYEIPPEK LVIYEQITRP LHEYKAIGPHVAVAKRLAAR GVKVRPGMVI GYIVLRGDGP ISKRAILAEEFDLRKHKYDA EYYIENQVLP AVLRILEAFG YRKEDLRWQK TXQTGLTAWL NIKKK;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 24K762Q variant of Deep Vent MILDADYITE DGKPIIRIFK KENGEFKVEY DRNFRPYIYADNA polymerase amino acid LLKDDSQIDE VRKITAERHG KIVRIIDAEK VRKKFLGRPIsequence EVWRLYFEHP QDVPAIRDKI REHSAVIDIF EYDIPFAKRYLIDKGLIPME GDEELKLLAF DIETLYHEGE EFAKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKVIREKDPDVIITYN GDSFDLPYLV KRAEKLGIKL PLGRDGSEPKMQRLGDMTAV EIKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YAHEIAEAWE TGKGLERVAK YSMEDAKVTYELGREFFPME AQLSRLVGQP LWDVSRSSTG NLVEWYLLRKAYERNELAPN KPDEREYERR LRESYAGGYV KEPEKGLWEGLVSLDFRSLY PSIIITHNVS PDTLNREGCR EYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQEIKRKMKA SKDPIEKKMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVRKELE EKFGFKVLYI DTDGLYATIP GAKPEEIKKKALEFVDYINA KLPGLLELEY EGFYVRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQAKVLEA ILKHGNVEEAVKIVKEVTEK LSKYEIPPEK LVIYEQITRP LHEYKAIGPHVAVAKRLAAR GVKVRPGMVI GYIVLRGDGP ISKRAILAEEFDLRKHKYDA EYYIENQVLP AVLRILEAFG YRKEDLRWQK TQQTGLTAWL NIKKK 25K762X variant of Deep Vent SYAGGYV KEPEKGLWEG LVSLDFRSLY PSIIITHNVSDNA polymerase catalytic PDTLNREGCR EYDVAPEVGH KFCKDFPGFI PSLLKRLLDEdomain amino acid sequence RQEIKRKMKA SKDPIEKKML DYRQRAIKIL ANSYYGYYGYAKARWYCKEC AESVTAWGRE YIEFVRKELE EKFGFKVLYIDTDGLYATIP GAKPEEIKKK ALEFVDYINA KLPGLLELEYEGFYVRGFFV TKKKYALIDE EGKIITRGLE IVRRDWSEIAKETQAKVLEA ILKHGNVEEA VKIVKEVTEK LSKYEIPPEKLVIYEQITRP LHEYKAIGPH VAVAKRLAAR GVKVRPGMVIGYIVLRGDGP ISKRAILAEE FDLRKHKYDA EYYIENQVLPAVLRILEAFG YRKEDLRWQK TXQTGL; wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 26K762Q variant of Deep Vent SYAGGYV KEPEKGLWEG LVSLDFRSLY PSIIITHNVSDNA polymerase catalytic PDTLNREGCR EYDVAPEVGH KFCKDFPGFI PSLLKRLLDEdomain amino acid sequence RQEIKRKMKA SKDPIEKKML DYRQRAIKIL ANSYYGYYGYAKARWYCKEC AESVTAWGRE YIEFVRKELE EKFGFKVLYIDTDGLYATIP GAKPEEIKKK ALEFVDYINA KLPGLLELEYEGFYVRGFFV TKKKYALIDE EGKIITRGLE IVRRDWSEIAKETQAKVLEA ILKHGNVEEA VKIVKEVTEK LSKYEIPPEKLVIYEQITRP LHEYKAIGPH VAVAKRLAAR GVKVRPGMVIGYIVLRGDGP ISKRAILAEE FDLRKHKYDA EYYIENQVLP AVLRILEAFG YRKEDLRWQK TQQTGL27 K762X variant of Deep VentMILDADYITE DGKPIIRIFK KENGEFKVEY DRNFRPYIYA DNA polymerase amino acidLLKDDSQIDE VRKITAERHG KIVRIIDAEK VRKKFLGRPI sequence with DNA bindingEVWRLYFEHP QDVPAIRDKI REHSAVIDIF EYDIPFAKRY domainLIDKGLIPME GDEELKLLAF DIETLYHEGE EFAKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKVIREKDPDVIITYN GDSFDLPYLV KRAEKLGIKL PLGRDGSEPKMQRLGDMTAV EIKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YAHEIAEAWE TGKGLERVAK YSMEDAKVTYELGREFFPME AQLSRLVGQP LWDVSRSSTG NLVEWYLLRKAYERNELAPN KPDEREYERR LRESYAGGYV KEPEKGLWEGLVSLDFRSLY PSIIITHNVS PDTLNREGCR EYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQEIKRKMKA SKDPIEKKMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVRKELE EKFGFKVLYI DTDGLYATIP GAKPEEIKKKALEFVDYINA KLPGLLELEY EGFYVRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQAKVLEA ILKHGNVEEAVKIVKEVTEK LSKYEIPPEK LVIYEQITRP LHEYKAIGPHVAVAKRLAAR GVKVRPGMVI GYIVLRGDGP ISKRAILAEEFDLRKHKYDA EYYIENQVLP AVLRILEAFG YRKEDLRWQKTXQTGLTAWL NIKKKGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 28K762Q variant of Deep Vent MILDADYITE DGKPIIRIFK KENGEFKVEY DRNFRPYIYADNA polymerase amino acid LLKDDSQIDE VRKITAERHG KIVRIIDAEK VRKKFLGRPIsequence with DNA binding EVWRLYFEHP QDVPAIRDKI REHSAVIDIF EYDIPFAKRYdomain LIDKGLIPME GDEELKLLAF DIETLYHEGE EFAKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKVIREKDPDVIITYN GDSFDLPYLV KRAEKLGIKL PLGRDGSEPKMQRLGDMTAV EIKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YAHEIAEAWE TGKGLERVAK YSMEDAKVTYELGREFFPME AQLSRLVGQP LWDVSRSSTG NLVEWYLLRKAYERNELAPN KPDEREYERR LRESYAGGYV KEPEKGLWEGLVSLDFRSLY PSIIITHNVS PDTLNREGCR EYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQEIKRKMKA SKDPIEKKMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVRKELE EKFGFKVLYI DTDGLYATIP GAKPEEIKKKALEFVDYINA KLPGLLELEY EGFYVRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQAKVLEA ILKHGNVEEAVKIVKEVTEK LSKYEIPPEK LVIYEQITRP LHEYKAIGPHVAVAKRLAAR GVKVRPGMVI GYIVLRGDGP ISKRAILAEEFDLRKHKYDA EYYIENQVLP AVLRILEAFG YRKEDLRWQKTQQTGLTAWL NIKKKGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 29K762X K775S variant of Deep MILDADYITE DGKPIIRIFK KENGEFKVEY DRNFRPYIYAVent DNA polymerase amino LLKDDSQIDE VRKITAERHG KIVRIIDAEK VRKKFLGRPIacid sequence with sequence EVWRLYFEHP QDVPAIRDKI REHSAVIDIF EYDIPFAKRYnon-specific DNA binding LIDKGLIPME GDEELKLLAF DIETLYHEGE EFAKGPIIMIdomain SYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKVIREKDPDVIITYN GDSFDLPYLV KRAEKLGIKL PLGRDGSEPKMQRLGDMTAV EIKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YAHEIAEAWE TGKGLERVAK YSMEDAKVTYELGREFFPME AQLSRLVGQP LWDVSRSSTG NLVEWYLLRKAYERNELAPN KPDEREYERR LRESYAGGYV KEPEKGLWEGLVSLDFRSLY PSIIITHNVS PDTLNREGCR EYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQEIKRKMKA SKDPIEKKMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVRKELE EKFGFKVLYI DTDGLYATIP GAKPEEIKKKALEFVDYINA KLPGLLELEY EGFYVRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQAKVLEA ILKHGNVEEAVKIVKEVTEK LSKYEIPPEK LVIYEQITRP LHEYKAIGPHVAVAKRLAAR GVKVRPGMVI GYIVLRGDGP ISKRAILAEEFDLRKHKYDA EYYIENQVLP AVLRILEAFG YRKEDLRWQKTXQTGLTAWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 30K762Q K775S variant of Deep MILDADYITE DGKPIIRIFK KENGEFKVEY DRNFRPYIYAVent DNA polymerase amino LLKDDSQIDE VRKITAERHG KIVRIIDAEK VRKKFLGRPIacid sequence with sequence EVWRLYFEHP QDVPAIRDKI REHSAVIDIF EYDIPFAKRYnon-specific DNA binding LIDKGLIPME GDEELKLLAF DIETLYHEGE EFAKGPIIMIdomain SYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKVIREKDPDVIITYN GDSFDLPYLV KRAEKLGIKL PLGRDGSEPKMQRLGDMTAV EIKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YAHEIAEAWE TGKGLERVAK YSMEDAKVTYELGREFFPME AQLSRLVGQP LWDVSRSSTG NLVEWYLLRKAYERNELAPN KPDEREYERR LRESYAGGYV KEPEKGLWEGLVSLDFRSLY PSIIITHNVS PDTLNREGCR EYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQEIKRKMKA SKDPIEKKMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVRKELE EKFGFKVLYI DTDGLYATIP GAKPEEIKKKALEFVDYINA KLPGLLELEY EGFYVRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQAKVLEA ILKHGNVEEAVKIVKEVTEK LSKYEIPPEK LVIYEQITRP LHEYKAIGPHVAVAKRLAAR GVKVRPGMVI GYIVLRGDGP ISKRAILAEEFDLRKHKYDA EYYIENQVLP AVLRILEAFG YRKEDLRWQKTQQTGLTAWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 31K764X variant of ThermococcusMILDTDYITK DGKPIIRIFK KENGEFKIEL DPHFQPYIYA litoralis DNA polymeraseLLKDDSAIEE IKAIKGERHG KTVRVLDAVK VRKKFLGREVEVWKLIFEHP QDVPAMRGKI REHPAVVDIY EYDIPFAKRYLIDKGLIPME GDEELKLLAF DIETFYHEGD EFGKGEIIMISYADEEEARV ITWKNIDLPY VDVVSNEREM IKRFVQVVKEKDPDVIITYN GDNFDLPYLI KRAEKLGVRL VLGRDKEHPEPKIQRMGDSF AVEIKGRIHF DLFPVVRRTI NLPTYTLEAVYEAVLGKTKS KLGAEEIAAI WETEESMKKL AQYSMEDARATYELGKEFFP MEAELAKLIG QSVWDVSRSS TGNLVEWYLLRVAYARNELA PNKPDEEEYK RRLRTTYLGG YVKEPEKGLWENIIYLDFRS LYPSIIVTHN VSPDTLEKEG CKNYDVAPIVGYRFCKDFPG FIPSILGDLI AMRQDIKKKM KSTIDPIEKKMLDYRQRAIK LLANSYYGYM GYPKARWYSK ECAESVTAWGRHYIEMTIRE IEEKFGFKVL YADTDGFYAT IPGEKPELIKKKAKEFLNYI NSKLPGLLEL EYEGFYLRGF FVTKKRYAVIDEEGRITTRG LEVVRRDWSE IAKETQAKVL EAILKEGSVEKAVEVVRDVV EKIAKYRVPL EKLVIHEQIT RDLKDYKAIGPHVAIAKRLA ARGIKVKPGT IISYIVLKGS GKISDRVILLTEYDPRKHKY DPDYYIENQV LPAVLRILEA FGYRKEDLRY QSSXQTGLDA WLKR;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 32K764Q variant of ThermococcusMILDTDYITK DGKPIIRIFK KENGEFKIEL DPHFQPYIYA litoralis DNA polymeraseLLKDDSAIEE IKAIKGERHG KTVRVLDAVK VRKKFLGREVEVWKLIFEHP QDVPAMRGKI REHPAVVDIY EYDIPFAKRYLIDKGLIPME GDEELKLLAF DIETFYHEGD EFGKGEIIMISYADEEEARV ITWKNIDLPY VDVVSNEREM IKRFVQVVKEKDPDVIITYN GDNFDLPYLI KRAEKLGVRL VLGRDKEHPEPKIQRMGDSF AVEIKGRIHF DLFPVVRRTI NLPTYTLEAVYEAVLGKTKS KLGAEEIAAI WETEESMKKL AQYSMEDARATYELGKEFFP MEAELAKLIG QSVWDVSRSS TGNLVEWYLLRVAYARNELA PNKPDEEEYK RRLRTTYLGG YVKEPEKGLWENIIYLDFRS LYPSIIVTHN VSPDTLEKEG CKNYDVAPIVGYRFCKDFPG FIPSILGDLI AMRQDIKKKM KSTIDPIEKKMLDYRQRAIK LLANSYYGYM GYPKARWYSK ECAESVTAWGRHYIEMTIRE IEEKFGFKVL YADTDGFYAT IPGEKPELIKKKAKEFLNYI NSKLPGLLEL EYEGFYLRGF FVTKKRYAVIDEEGRITTRG LEVVRRDWSE IAKETQAKVL EAILKEGSVEKAVEVVRDVV EKIAKYRVPL EKLVIHEQIT RDLKDYKAIGPHVAIAKRLA ARGIKVKPGT IISYIVLKGS GKISDRVILLTEYDPRKHKY DPDYYIENQV LPAVLRILEA FGYRKEDLRY QSSQQTGLDA WLKR 33K764X variant of Thermococcus TYLGG YVKEPEKGLW ENIIYLDFRS LYPSIIVTHNlitoralis DNA polymerase VSPDTLEKEG CKNYDVAPIV GYRFCKDFPG FIPSILGDLIcatalytic domain amino acid AMRQDIKKKM KSTIDPIEKK MLDYRQRAIK LLANSYYGYMsequence GYPKARWYSK ECAESVTAWG RHYIEMTIRE IEEKFGFKVLYADTDGFYAT IPGEKPELIK KKAKEFLNYI NSKLPGLLELEYEGFYLRGF FVTKKRYAVI DEEGRITTRG LEVVRRDWSEIAKETQAKVL EAILKEGSVE KAVEVVRDVV EKIAKYRVPLEKLVIHEQIT RDLKDYKAIG PHVAIAKRLA ARGIKVKPGTIISYIVLKGS GKISDRVILL TEYDPRKHKY DPDYYIENQVLPAVLRILEA FGYRKEDLRY QSSXQTGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 34K764Q variant of Thermococcus TYLGG YVKEPEKGLW ENIIYLDFRS LYPSIIVTHNlitoralis DNA polymerase VSPDTLEKEG CKNYDVAPIV GYRFCKDFPG FIPSILGDLIcatalytic domain amino acid AMRQDIKKKM KSTIDPIEKK MLDYRQRAIK LLANSYYGYMsequence GYPKARWYSK ECAESVTAWG RHYIEMTIRE IEEKFGFKVLYADTDGFYAT IPGEKPELIK KKAKEFLNYI NSKLPGLLELEYEGFYLRGF FVTKKRYAVI DEEGRITTRG LEVVRRDWSEIAKETQAKVL EAILKEGSVE KAVEVVRDVV EKIAKYRVPLEKLVIHEQIT RDLKDYKAIG PHVAIAKRLA ARGIKVKPGTIISYIVLKGS GKISDRVILL TEYDPRKHKY DPDYYIENQVLPAVLRILEA FGYRKEDLRY QSSQQTGL 35 K764X variant of ThermococcusMILDTDYITK DGKPIIRIFK KENGEFKIEL DPHFQPYIYA litoralis DNA polymerase,LLKDDSAIEE IKAIKGERHG KSVRVVDAVK VKKKFLGREV sequence 2 (acc. ADK47977.1)EVWKLIFEHP QDVPAMRDKI KEHPAVIDIY EYDIPFAKRYLIDKGLIPME GDEELKLLAF DIETFYHEGD EFGKGEIIMISYADEEEARV ITWKNIDLPY VDVVSNEREM IKRFVQVVKEKDPDVIITYN GDNFDLPYLI KRAEKLGVRL VLGRDKENPEPKIQRMGDSF AVEIKGRIHF DLFPVVRRTI NLPTYTLEAVYEAVLGKTKS KLGAEEIAAI WETEESMKKL AQYSMEDARATYELGKEFFP MEAELAKLIG QSVWDVSRSS TGNLVEWYLLRVAYERNELA PNKPDEEEYK RRLRTTYLGG YVKEPEKGLWENIIYLDFRS LYPSIIVTHN VSPDTLEKEG CENYDIAPIVSYRFCKDFPG FIPSILGDLI AMRQEIKKKM KATIDPVERKMLDYRQRAVK LLANSYYGYM GYPKARWYSK ECAESVTAWGRHYIEMTIKE IEEKFGFKVL YADTDGFYAT ISGEKPEIIKKKAREFLNYI NSKLPGLLEL EYEGFYLRGF FVTKKRYAVIDEEGRITTRG LEVVRRDWSE IAKETQAKVL EAILKDGSVEKAVEIVRDVL EKIAKYRVPL EKLVIHEQIT RDLKDYKAIGPHVAIAKRLA ARGIKVKPGT IISYIVLKGS GKISDRVILLTEYDPEKHKY DPDYYIENQV LPAVLRILEA FGYRKEDLRY QSSXQTGLDA WLKR;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 36K764Q variant of ThermococcusMILDTDYITK DGKPIIRIFK KENGEFKIEL DPHFQPYIYA litoralis DNA polymerase,LLKDDSAIEE IKAIKGERHG KSVRVVDAVK VKKKFLGREV sequence 2 (acc. ADK47977.1)EVWKLIFEHP QDVPAMRDKI KEHPAVIDIY EYDIPFAKRYLIDKGLIPME GDEELKLLAF DIETFYHEGD EFGKGEIIMISYADEEEARV ITWKNIDLPY VDVVSNEREM IKRFVQVVKEKDPDVIITYN GDNFDLPYLI KRAEKLGVRL VLGRDKENPEPKIQRMGDSF AVEIKGRIHF DLFPVVRRTI NLPTYTLEAVYEAVLGKTKS KLGAEEIAAI WETEESMKKL AQYSMEDARATYELGKEFFP MEAELAKLIG QSVWDVSRSS TGNLVEWYLLRVAYERNELA PNKPDEEEYK RRLRTTYLGG YVKEPEKGLWENIIYLDFRS LYPSIIVTHN VSPDTLEKEG CENYDIAPIVSYRFCKDFPG FIPSILGDLI AMRQEIKKKM KATIDPVERKMLDYRQRAVK LLANSYYGYM GYPKARWYSK ECAESVTAWGRHYIEMTIKE IEEKFGFKVL YADTDGFYAT ISGEKPEIIKKKAREFLNYI NSKLPGLLEL EYEGFYLRGF FVTKKRYAVIDEEGRITTRG LEVVRRDWSE IAKETQAKVL EAILKDGSVEKAVEIVRDVL EKIAKYRVPL EKLVIHEQIT RDLKDYKAIGPHVAIAKRLA ARGIKVKPGT IISYIVLKGS GKISDRVILLTEYDPEKHKY DPDYYIENQV LPAVLRILEA FGYRKEDLRY QSSQQTGLDA WLKR 37K764X variant of Thermococcus TYLGG YVKEPEKGLW ENIIYLDFRS LYPSIIVTHNlitoralis DNA polymerase, VSPDTLEKEG CENYDIAPIV SYRFCKDFPG FIPSILGDLIsequence 2 (acc. ADK47977.1),AMRQEIKKKM KATIDPVERK MLDYRQRAVK LLANSYYGYM catalytic domain amino acidGYPKARWYSK ECAESVTAWG RHYIEMTIKE IEEKFGFKVL sequenceYADTDGFYAT ISGEKPEIIK KKAREFLNYI NSKLPGLLELEYEGFYLRGF FVTKKRYAVI DEEGRITTRG LEVVRRDWSETAKETQAKVL EAILKDGSVE KAVEIVRDVL EKIAKYRVPLEKLVIHEQIT RDLKDYKAIG PHVAIAKRLA ARGIKVKPGTIISYIVLKGS GKISDRVILL TEYDPEKHKY DPDYYIENQVLPAVLRILEA FGYRKEDLRY QSSXQTGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 38K764Q variant of Thermococcus TYLGG YVKEPEKGLW ENIIYLDFRS LYPSIIVTHNlitoralis DNA polymerase, VSPDTLEKEG CENYDIAPIV SYRFCKDFPG FIPSILGDLIsequence 2 (acc. ADK47977.1),AMRQEIKKKM KATIDPVERK MLDYRQRAVK LLANSYYGYM catalytic domain amino acidGYPKARWYSK ECAESVTAWG RHYIEMTIKE IEEKFGFKVL sequenceYADTDGFYAT ISGEKPEIIK KKAREFLNYI NSKLPGLLELEYEGFYLRGF FVTKKRYAVI DEEGRITTRG LEVVRRDWSETAKETQAKVL EAILKDGSVE KAVEIVRDVL EKIAKYRVPLEKLVIHEQIT RDLKDYKAIG PHVAIAKRLA ARGIKVKPGTIISYIVLKGS GKISDRVILL TEYDPEKHKY DPDYYIENQVLPAVLRILEA FGYRKEDLRY QSSQQTGL 39 R761X variant of ThermococcusMILDTDYITE DGKPVIRIFK KENGEFKIDY DRNFEPYIYA gorgonarius DNA polymeraseLLKDDSAIED VKKITAERHG TTVRVVRAEK VKKKFLGRPIEVWKLYFTHP QDVPAIRDKI KEHPAVVDIY EYDIPFAKRYLIDKGLIPME GDEELKMLAF DIETLYHEGE EFAEGPILMISYADEEGARV ITWKNIDLPY VDVVSTEKEM IKRFLKVVKEKDPDVLITYN GDNFDFAYLK KRSEKLGVKF ILGREGSEPKIQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYEAIFGQPKEKV YAEEIAQAWE TGEGLERVAR YSMEDAKVTYELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDERELARR RESYAGGYVK EPERGLWENIVYLDFRSLYP SIIITHNVSP DTLNREGCEE YDVAPQVGHKFCKDFPGFIP SLLGDLLEER QKVKKKMKAT IDPIEKKLLDYRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGRQYIETTIREIEE KFGFKVLYAD TDGFFATIPG ADAETVKKKAKEFLDYINAK LPGLLELEYE GFYKRGFFVT KKKYAVIDEEDKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAVRIVKEVTEKL SKYEVPPEKL VIYEQITRDL KDYKATGPHVAVAKRLAARG IKIRPGTVIS YIVLKGSGRI GDRAIPFDEFDPAKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT XQVGLGAWLK PKT;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 40R761Q variant of ThermococcusMILDTDYITE DGKPVIRIFK KENGEFKIDY DRNFEPYIYA gorgonarius DNA polymeraseLLKDDSAIED VKKITAERHG TTVRVVRAEK VKKKFLGRPIEVWKLYFTHP QDVPAIRDKI KEHPAVVDIY EYDIPFAKRYLIDKGLIPME GDEELKMLAF DIETLYHEGE EFAEGPILMISYADEEGARV ITWKNIDLPY VDVVSTEKEM IKRFLKVVKEKDPDVLITYN GDNFDFAYLK KRSEKLGVKF ILGREGSEPKIQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYEAIFGQPKEKV YAEEIAQAWE TGEGLERVAR YSMEDAKVTYELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDERELARR RESYAGGYVK EPERGLWENIVYLDFRSLYP SIIITHNVSP DTLNREGCEE YDVAPQVGHKFCKDFPGFIP SLLGDLLEER QKVKKKMKAT IDPIEKKLLDYRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGRQYIETTIREIEE KFGFKVLYAD TDGFFATIPG ADAETVKKKAKEFLDYINAK LPGLLELEYE GFYKRGFFVT KKKYAVIDEEDKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAVRIVKEVTEKL SKYEVPPEKL VIYEQITRDL KDYKATGPHVAVAKRLAARG IKIRPGTVIS YIVLKGSGRI GDRAIPFDEFDPAKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT QQVGLGAWLK PKT 41R761X variant of Thermococcus SYAGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSPgorgonarius DNA polymerase, DTLNREGCEE YDVAPQVGHK FCKDFPGFIP SLLGDLLEERcatalytic domain amino acid QKVKKKMKAT IDPIEKKLLD YRQRAIKILA NSFYGYYGYAsequence KARWYCKECA ESVTAWGRQY IETTIREIEE KFGFKVLYADTDGFFATIPG ADAETVKKKA KEFLDYINAK LPGLLELEYEGFYKRGFFVT KKKYAVIDEE DKITTRGLEI VRRDWSEIAKETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKLVIYEQITRDL KDYKATGPHV AVAKRLAARG IKIRPGTVISYIVLKGSGRI GDRAIPFDEF DPAKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT XQVGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 42R761Q variant of Thermococcus SYAGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSPgorgonarius DNA polymerase, DTLNREGCEE YDVAPQVGHK FCKDFPGFIP SLLGDLLEERcatalytic domain amino acid QKVKKKMKAT IDPIEKKLLD YRQRAIKILA NSFYGYYGYAsequence KARWYCKECA ESVTAWGRQY IETTIREIEE KFGFKVLYADTDGFFATIPG ADAETVKKKA KEFLDYINAK LPGLLELEYEGFYKRGFFVT KKKYAVIDEE DKITTRGLEI VRRDWSEIAKETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKLVIYEQITRDL KDYKATGPHV AVAKRLAARG IKIRPGTVISYIVLKGSGRI GDRAIPFDEF DPAKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT QQVGL43 R761X variant of ThermococcusMILDTDYITE DGKPVIRIFK KENGEFKIEY DRTFEPYFYA kodakarensis DNA polymeraseLLKDDSAIEE VKKITAERHG TVVTVKRVEK VQKKFLGRPVEVWKLYFTHP QDVPAIRDKI REHPAVIDIY EYDIPFAKRYLIDKGLVPME GDEELKMLAF DIETLYHEGE EFAEGPILMISYADEEGARV ITWKNVDLPY VDVVSTEREM IKRFLRVVKEKDPDVLITYN GDNFDFAYLK KRCEKLGINF ALGRDGSEPKIQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYEAVFGQPKEKV YAEEITTAWE TGENLERVAR YSMEDAKVTYELGKEFLPME AQLSRLIGQS LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEKELARR RQSYEGGYVK EPERGLWENIVYLDFRSLYP SIIITHNVSP DTLNREGCKE YDVAPQVGHRFCKDFPGFIP SLLGDLLEER QKIKKKMKAT IDPIERKLLDYRQRAIKILA NSYYGYYGYA RARWYCKECA ESVTAWGREYITMTIKEIEE KYGFKVIYSD TDGFFATIPG ADAETVKKKAMEFLKYINAK LPGALELEYE GFYKRGFFVT KKKYAVIDEEGKITTRGLEI VRRDWSEIAK ETQARVLEAL LKDGDVEKAVRIVKEVTEKL SKYEVPPEKL VIHEQITRDL KDYKATGPHVAVAKRLAARG VKIRPGTVIS YIVLKGSGRI GDRAIPFDEFDPTKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT XQVGLSAWLK PKGT;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 44R761Q variant of ThermococcusMILDTDYITE DGKPVIRIFK KENGEFKIEY DRTFEPYFYA kodakarensis DNA polymeraseLLKDDSAIEE VKKITAERHG TVVTVKRVEK VQKKFLGRPVEVWKLYFTHP QDVPAIRDKI REHPAVIDIY EYDIPFAKRYLIDKGLVPME GDEELKMLAF DIETLYHEGE EFAEGPILMISYADEEGARV ITWKNVDLPY VDVVSTEREM IKRFLRVVKEKDPDVLITYN GDNFDFAYLK KRCEKLGINF ALGRDGSEPKIQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYEAVFGQPKEKV YAEEITTAWE TGENLERVAR YSMEDAKVTYELGKEFLPME AQLSRLIGQS LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEKELARR RQSYEGGYVK EPERGLWENIVYLDFRSLYP SIIITHNVSP DTLNREGCKE YDVAPQVGHRFCKDFPGFIP SLLGDLLEER QKIKKKMKAT IDPIERKLLDYRQRAIKILA NSYYGYYGYA RARWYCKECA ESVTAWGREYITMTIKEIEE KYGFKVIYSD TDGFFATIPG ADAETVKKKAMEFLKYINAK LPGALELEYE GFYKRGFFVT KKKYAVIDEEGKITTRGLEI VRRDWSEIAK ETQARVLEAL LKDGDVEKAVRIVKEVTEKL SKYEVPPEKL VIHEQITRDL KDYKATGPHVAVAKRLAARG VKIRPGTVIS YIVLKGSGRI GDRAIPFDEFDPTKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT QQVGLSAWLK PKGT 45R761X variant of Thermococcus SYEGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSPkodakarensis DNA polymerase, DTLNREGCKE YDVAPQVGHR FCKDFPGFIP SLLGDLLEERcatalytic domain amino acid QKIKKKMKAT IDPIERKLLD YRQRAIKILA NSYYGYYGYAsequence RARWYCKECA ESVTAWGREY ITMTIKEIEE KYGFKVIYSDTDGFFATIPG ADAETVKKKA MEFLKYINAK LPGALELEYEGFYKRGFFVT KKKYAVIDEE GKITTRGLEI VRRDWSEIAKETQARVLEAL LKDGDVEKAV RIVKEVTEKL SKYEVPPEKLVIHEQITRDL KDYKATGPHV AVAKRLAARG VKIRPGTVISYIVLKGSGRI GDRAIPFDEF DPTKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT XQVGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 46R761Q variant of Thermococcus SYEGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSPkodakarensis DNA polymerase, DTLNREGCKE YDVAPQVGHR FCKDFPGFIP SLLGDLLEERcatalytic domain amino acid QKIKKKMKAT IDPIERKLLD YRQRAIKILA NSYYGYYGYAsequence RARWYCKECA ESVTAWGREY ITMTIKEIEE KYGFKVIYSDTDGFFATIPG ADAETVKKKA MEFLKYINAK LPGALELEYEGFYKRGFFVT KKKYAVIDEE GKITTRGLEI VRRDWSEIAKETQARVLEAL LKDGDVEKAV RIVKEVTEKL SKYEVPPEKLVIHEQITRDL KDYKATGPHV AVAKRLAARG VKIRPGTVISYIVLKGSGRI GDRAIPFDEF DPTKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT QQVGL47 K761X variant of GYAGGYVK EPERGLWDNI VYLDFRSLYP SIIITHNVSPThermococcus species 9 N-7 DTLNREGCKE YDVAPEVGHK FCKDFPGFIP SLLGDLLEERDNA polymerase, catalytic QKIKRKMKAT VDPLEKKLLD YRQRAIKILA NSFYGYYGYAdomain amino acid sequence KARWYCKECA ESVTAWGREY IEMVIRELEE KFGFKVLYADTDGLHATIPG ADAETVKKKA KEFLKYINPK LPGLLELEYEGFYVRGFFVT KKKYAVIDEE GKITTRGLEI VRRDWSEIAKETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKLVIHEQITRDL RDYKATGPHV AVAKRLAARG VKIRPGTVISYIVLKGSGRI GDRAIPADEF DPTKHRYDAE YYIENQVLPA VERILKAFGY RKEDLRYQKT XQVGL;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 48 K761Q variant ofGYAGGYVK EPERGLWDNI VYLDFRSLYP SIIITHNVSP Thermococcus species 90 N-7DTLNREGCKE YDVAPEVGHK FCKDFPGFIP SLLGDLLEER DNA polymerase, catalyticQKIKRKMKAT VDPLEKKLLD YRQRAIKILA NSFYGYYGYA domain amino acid sequenceKARWYCKECA ESVTAWGREY IEMVIRELEE KFGFKVLYADTDGLHATIPG ADAETVKKKA KEFLKYINPK LPGLLELEYEGFYVRGFFVT KKKYAVIDEE GKITTRGLEI VRRDWSEIAKETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKLVIHEQITRDL RDYKATGPHV AVAKRLAARG VKIRPGTVISYIVLKGSGRI GDRAIPADEF DPTKHRYDAE YYIENQVLPA VERILKAFGY RKEDLRYQKT QQVGL49 K761X variant of MILDTDYITE NGKPVIRVFK KENGEFKIEY DRTFEPYFYAThermococcus species 90 N-7 LLKDDSAIED VKKVTAKRHG TVVKVKRAEK VQKKFLGRPIDNA polymerase EVWKLYFNHP QDVPAIRDRI RAHPAVVDIY EYDIPFAKRYLIDKGLIPME GDEELTMLAF DIETLYHEGE EFGTGPILMISYADGSEARV ITWKKIDLPY VDVVSTEKEM IKRFLRVVREKDPDVLITYN GDNFDFAYLK KRCEELGIKF TLGRDGSEPKIQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYEAVFGKPKEKV YAEEIAQAWE SGEGLERVAR YSMEDAKVTYELGREFFPME AQLSRLIGQS LWDVSRSSTG NLVEWFLLRKAYKRNELAPN KPDERELARR RGGYAGGYVK EPERGLWDNIVYLDFRSLYP SIIITHNVSP DTLNREGCKE YDVAPEVGHKFCKDFPGFIP SLLGDLLEER QKIKRKMKAT VDPLEKKLLDYRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGREYIEMVIRELEE KFGFKVLYAD TDGLHATIPG ADAETVKKKAKEFLKYINPK LPGLLELEYE GFYVRGFFVT KKKYAVIDEEGKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAVRIVKEVTEKL SKYEVPPEKL VIHEQITRDL RDYKATGPHVAVAKRLAARG VKIRPGTVIS YIVLKGSGRI GDRAIPADEFDPTKHRYDAE YYIENQVLPA VERILKAFGY RKEDLRYQKT XQVGLGAWLK VKGKK;wherein X is selected from Q, N, H, S, T,Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 50 K761Q variant ofMILDTDYITE NGKPVIRVFK KENGEFKIEY DRTFEPYFYA Thermococcus species 90 N-7LLKDDSAIED VKKVTAKRHG TVVKVKRAEK VQKKFLGRPI DNA polymeraseEVWKLYFNHP QDVPAIRDRI RAHPAVVDIY EYDIPFAKRYLIDKGLIPME GDEELTMLAF DIETLYHEGE EFGTGPILMISYADGSEARV ITWKKIDLPY VDVVSTEKEM IKRFLRVVREKDPDVLITYN GDNFDFAYLK KRCEELGIKF TLGRDGSEPKIQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYEAVFGKPKEKV YAEEIAQAWE SGEGLERVAR YSMEDAKVTYELGREFFPME AQLSRLIGQS LWDVSRSSTG NLVEWFLLRKAYKRNELAPN KPDERELARR RGGYAGGYVK EPERGLWDNIVYLDFRSLYP SIIITHNVSP DTLNREGCKE YDVAPEVGHKFCKDFPGFIP SLLGDLLEER QKIKRKMKAT VDPLEKKLLDYRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGREYIEMVIRELEE KFGFKVLYAD TDGLHATIPG ADAETVKKKAKEFLKYINPK LPGLLELEYE GFYVRGFFVT KKKYAVIDEEGKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAVRIVKEVTEKL SKYEVPPEKL VIHEQITRDL RDYKATGPHVAVAKRLAARG VKIRPGTVIS YIVLKGSGRI GDRAIPADEFDPTKHRYDAE YYIENQVLPA VERILKAFGY RKEDLRYQKT QQVGLGAWLK VKGKK 51E775Q variant of Pyrobaculum MRFWPLDATY SVVGGVPEVR VFGVDGEGRR VVLVDRRFRPcalidifontis DNA polymerase YFYAKCDKCD ASLAKSYLSR VAPVEAVEVV ERRFFGRPTIFLKVVAKVPE DVRKLREAAL GAPGVVDVYE ADIRYYMRYMIDKGVVPCAW NVVEAREAGK LGPLPLYEVV EWAGVEEGFPPPLRVLAFDI EVYNERGSPD PLRDPVVMLA VKTSDGREEVFEAEGRDDRR VIRGFVDFVK EFDPDVIVGY NSNGFDWPYLSERAKALGVP LRVDRLGGVP QQSVYGHWSV VGRANVDLYNIVDEFPEIKV KTLDRVAEYF GVMKRSERVL IPGHKVYEYWNDPAKRPTLM RYVLDDVRST LGLAEKLLPF LIQLSSVSGLPLDQVAAASV GNRVEWMLLR YAYRMGEVAP NREEREYEPYKGAIVLEPKP GLYSDVLVLD FSSMYPNIMM KYNLSPDTYLEPHEPDPPEG VVVAPEVGHR FRKAPTGFIP AVLKHLVELRRAVREEAKKY PPDSPEYRLL DERQRALKVM ANAMYGYLGWVGARWYKKEV AESVTAFARA ILLDVVEYAK RLGIEVIYGDTDSLFVKKSG AVDRLVKYVE ERHGIEIKVD KDYERVLFTEAKKRYAGLLR DGRIDIVGFE VVRGDWCELA KEVQLNVVELILKSKSVGEA RERVVKYVRE VVERLKAYKF DLDDLIIWKTLDKELDEYKA YGPHVHAALE LKRRGYKVGK GTTVGYVIVRGPGKVSERAM PYIFVDDASK VDVDYYIEKQ VIPAALRIAE VLGVKESDLK TGRVQKSLLD FLG 52E778Q variant of Pyrobaculum MKFKLWPLDA TYSVVGGVPE VRIFGISESG DRVVVVDRRFaerophilum DNA polymerase RPYFYADCPA CDPESVRSQL GRVAPVEEVV AVERRYLGRPRSFLKIVARV PEDVRKLREA AAALPGVSGV YEADIRFYMRYMLDMGVVPC SWNTVDAEAT GEKLGNLPVY KVAEWGGVTEGFPPPLRVLA FDIEVYNERG TPDPLRDPVI LLAVQASDGRVEVFEASGRD DRSVLRSFID FVREFDPDVI VGYNSNQFDWPYLAERARAL GIPLKVDRVG GAPQQSVYGH WSVTGRAMVDLYNIVDEFPE IKLKTLDRVA EYFGVMKREE RVLVPGHKIYEYWRDQGKRP LLRQYVIDDV KSTYGLAEKL LPFLIQLSSVSGLPLDQVAA ASVGNRVEWM LLRYAYRLGE VAPNREEREYEPYKGAIVLE PRPGLYSDVL ALDFSSMYPN IMMKYNLSPDTYLERGEPDP PGGVYVAPEV GHRFRREPPG FIPLVLRQLIELRKRVREEL KKYPPDSPEY RVLDERQRAL KIMANAMYGYTGWVGARWYK KEVAESVTAF ARAILKDVIE YARKAGIVVIYGDTDSLFVK KSGDVEKLVK YVEEKYGIDI KIDKDYSTVLFTEAKKRYAG LLRDGRIDIV GFEVVRGDWS ELAKEVQLRVIELILTSRDV SEARQKVVKY VRGVIDKLRN YEVDLDDLIIWKTLDKELDE YKAYPPHVHA AILLKKRGYK VGKGTTIGYVVVKGGEKVSE RAVPYIFIDD IEKIDLDYYV ERQVIPAALR IAEVIGIKEG DLKTGRSQRT LLDFF53 Sso7d SNS-dsDBD amino acidATVKFKYKGE EKEVDISKIK KVWRVGKMIS FTYDEGGGKT sequence of SulfolobusGRGAVSEKDA PKELLQMLEK QKK solfataricus (see U.S. Pat. No. 6,627,424) 54Sac7d SNS-dsDBD amino acid VKVKFKYKGE EKEVDTSKIK KVWRVGKMVS FTYDDNGKTGsequence of Sulfolobus RGAVSEKDAP KELLDMLARA EREKK acidocaldarius 55Pyrobaculum aerophilum SKKQKLKFYD IKAKQAFETD QYEVIEKQTA RGPMMFAVAKPae3192 amino acid sequence SPYTGIKVYR LLGKKK 56 Pyrobaculum aerophilumAKQKLKFYDI KAKQSFETDK YEVIEKETAR GPMLFAVATS Pae0384 amino acid sequencePYTGIKVYRL LGKKK 57 Aeropyrum pernix Ape3192PKKEKIKFFD LVAKKYYETD NYEVEIKETK RGKFRFAKAK amino acid sequenceSPYTGKIFYR VLGKA 58 HMfA HMf family archaealGELPIAPIGR IIKNAGAERV SDDARIALAK VLEEMGEEIAhistone amino acid sequence of SEAVKLAKHA GRKTIKAEDMethanothermus fervidus 59 HMfB HMf family archaealELPIAPIGRI IKDAGAERVS DDARITLAKI LEEMGRDIAShistone amino acid sequence of EAIKLARHAG RKTIKAEDIMethanothermus fervidus 60 HpyA1 HMf family archaealGELPIAPVDR LIRKAGAERV SEEAAKILAE YLEEYAIEVShistone amino acid sequence of KKAVEFARHA GRKTVKAEDPyrococcus strain GB-3a 61 HpyA2 HMf family archaealAELPIAPVDR LIRKAGAQRV SEQAAKLLAE HLEEKALEIAhistone amino acid sequence of RKAVDLAKHA GRKTVKAEDPyrococcus strain GB-3a 62 Sso7d sequence non-specificATVKFKYKGE EKEVDISKIK KVWRVGKMIS FTYDEGGGKTDNA-binding domain amino acid GRGAVSEKDA PKELLQMLEK QK sequence 63Pyrococcus 3′-5′ exonuclease EELKLLAFDI ETLYHEGEEF GKGPIIMISY ADEEEAKVITdomain amino acid sequence WKKIDLPYVE VVSSEREMIK RFLKIIREKD PDIIITYNGDSFDLPYLAKR AEKLGIKLTI GRDGSEPKMQ RIGDMTAVEVKGRIHFDLYH VIRRTINLPT YTLEAVYEAI FGKPKEKVYADEIAKAWETG EGLERVAKYS MEDAKATYEL GKEF 64 PCR Primer nucleotide sequenceGAAGAGCCAAGGACAGGTAC 65 PCR Primer nucleotide sequenceCCTCCAAATCAAGCCTCTAC 66 PCR Primer nucleotide sequenceCAGTGCAGTGCTTGATAACAGG 67 PCR Primer nucleotide sequenceGTAGTGCGCGTTTGATTTCC 68 PCR Primer nucleotide sequenceCCTGCTCTGCCGCTTCACGC 69 PCR Primer nucleotide sequenceCGAACGTCGCGCAGAGAAACAGG 70 PCR Primer nucleotide sequenceCTGATGAGTTCGTGTCCGTACAACTGGCGTAATC 71 PCR Primer nucleotide sequenceGTGCACCATGCAACATGAATAACAGTGGGTTATC 72 PCR Primer nucleotide sequenceGGGCGTTTTCCGTAACACTG 73 PCR Primer nucleotide sequenceTGACCACATACAATCGCCGT 74 PCR Primer nucleotide sequenceCTCCACAGGGTGAGGTCTAAGTGATGACA 75 PCR Primer nucleotide sequenceCAATCTCAGGGCAAGTTAAGGGAATAGTG

What is claimed is:
 1. A thermophilic DNA polymerase comprising a familyB polymerase catalytic domain, the family B polymerase catalytic domainhaving an amino acid sequence in which the position corresponding toposition 379 of SEQ ID NO: 6 is a neutral amino acid residue selectedfrom Q, N, H, S, T, Y, C, W, A, F, P and G, wherein the family Bpolymerase catalytic domain has at least 90%, identity to the family Bpolymerase catalytic domain sequence of a sequence selected from SEQ IDNOs: 15, 25, 33, 41 and 47, wherein said therm ophilic DNA polymerasepossesses increased inhibitor tolerance and/or increased yield comparedto a thermophilic DNA polymerase in which the position corresponding toposition 379 of SEQ ID NO: 6 is not a neutral amino acid.
 2. Thethermophilic DNA polymerase of claim 1, wherein the family B polymerasecatalytic domain has at least 80%, identity to SEQ ID NO:
 6. 3. Thethermophilic DNA polymerase of claim 1, wherein the amino acid residueat the position of the amino acid sequence that corresponds to position25 of SEQ ID NO: 6 is a serine.
 4. The thermophilic DNA polymerase claim1, wherein the family B polymerase catalytic domain comprises aconsecutive amino acid sequence of WQKTX, XQTGL, KTXQT, YQKTX, XQVGL,KTXQV, YQSSX, XQTGL, SSXQT, TGRVX, XKSLL, RVXKS, TGRSX, XRTLL, or RSXRT;wherein X is a neutral amino acid residue; and wherein X is within 20residues of the C-terminus of the family B polymerase catalytic domain.5. The thermophilic DNA polymerase of claim 1, which comprises asequence non-specific double-stranded DNA-binding domain.
 6. Thethermophilic DNA polymerase of claim 5, wherein the sequencenon-specific double-stranded DNA-binding domain comprises an amino acidsequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identity to a sequence selected from SEQ ID NOs: 53 to
 62. 7. Thethermophilic DNA polymerase of claim 5, wherein the sequencenon-specific double-stranded DNA-binding domain is C-terminal to thefamily B polymerase catalytic domain.
 8. The thermophilic DNA polymeraseof claim 5, wherein the sequence non-specific double-strandedDNA-binding domain is an Sso7d, Sac7d, or Sac7e domain.
 9. Thethermophilic DNA polymerase of claim 1, wherein the thermophilic DNApolymerase comprises: (a) the consecutive amino acid residues LDFRS, (b)the consecutive amino acid residues FRSLY, or(c) the consecutive aminoacid residues SLYPS, wherein the serine residues in LDFRS and FRSLY, andthe first serine residue in SLYPS within 30 amino acid residues of theN-terminus of the family B polymerase catalytic domain.
 10. Thethermophilic DNA polymerase of claim 1, wherein the neutral amino acidresidue is a polar neutral amino acid residue.
 11. The thermophilic DNApolymerase of claim 1, wherein the neutral amino acid residue comprisesan amide.
 12. The thermophilic DNA polymerase of claim 1, wherein theneutral amino acid residue is selected from Q and N.
 13. Thethermophilic DNA polymerase of claim 1, which comprises a 3′ to 5′exonuclease domain.
 14. The thermophilic DNA polymerase of claim 13,wherein the 3′ to 5′ exonuclease domain comprises an amino acid sequencehaving at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 63.15. A method of in vitro nucleic acid synthesis comprising contacting atleast one primer and at least one template with the thermophilic DNApolymerase claim 1 in the presence of at least one dNTP.
 16. The methodof claim 15, wherein the thermophilic DNA polymerase is initially boundto a thermolabile inhibitor and the method comprises denaturing theinhibitor.
 17. The method of claim 15, further comprising amplificationof the template.
 18. The method of claim 17, wherein the amplificationcomprises PCR.